FRESH CUT FRUITS AND VEGETABLES...Knowledge of the nature of fresh-cut fruits and vegetables as they...

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Science, Technology, and Market FRESH- CUT FRUITS AND VEGETABLES © 2002 by CRC Press LLC

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Science, Technology, and Market

FRESH-CUT FRUITSAND VEGETABLES

© 2002 by CRC Press LLC

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CRC PR ESSBoca Raton London New York Washington, D.C.

Science, Technology, and Market

Edited by Olusola Lamikanra

FRESH-CUT FRUITSAND VEGETABLES

© 2002 by CRC Press LLC

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This book contains information obtained from authentic and highly regarded sources. Reprinted materialis quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonableefforts have been made to publish reliable data and information, but the authors and the publisher cannotassume responsibility for the validity of all materials or for the consequences of their use.

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No claim to original U.S. Government worksInternational Standard Book Number 1-58716-030-7

Library of Congress Card Number 2001054766Printed in the United States of America 1 2 3 4 5 6 7 8 9 0

Printed on acid-free paper

Library of Congress Cataloging-in-Publication Data

Fresh-cut fruits and vegetables: science, technology, and market /edited by Olusola Lamikanra.

p. cm.Includes bibliographical references and index.ISBN 1-58716-030-7 (alk. paper)1. Fruit–Analysis. 2. Fruit–Preservation. 3. Vegetables–Analysis. 4. Vegetables–

Preservation. I. Lamikanra, Olusola.TX612.F7 F74 2002664

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Preface

Fresh-cut fruits and vegetables are a relatively new and rapidly developing segmentof the fresh produce industry. Fresh-cut products have been freshly cut, washed,packaged, and maintained with refrigeration. They are in a raw state and even thoughminimally processed, they remain in a fresh state, ready to eat or cook. The Interna-tional Fresh-cut Produce Association (IFPA) defines fresh-cut products as fruits orvegetables that have been trimmed and/or peeled and/or cut into 100% usable productthat is bagged or prepackaged to offer consumers high nutrition, convenience, andflavor while still maintaining its freshness. Industry estimates in the U.S. indicate thatfresh-cut sales of approximately $11 billion in 2000 account for over 10% of the totalfresh fruit and vegetable market, with food service sales making up 60% of the total.Sales are projected to increase by 10–15% annually for the next five years.

High levels of quality accompanied by superior safety are essential for sustainedindustry growth and fresh-cut produce consumption. Fresh-cut fruit and vegetableproducts differ from traditional, intact fruit and vegetables in terms of their physi-ology, handling and storage requirements. The disruption of tissue and cell integritythat result from fresh-cut processing decreases produce product shelf life. Conse-quently, fresh-cut products require very special attention because of the magnitudeof enzymatic and respiratory factors as well as microbiological concerns that impacton safety.

Knowledge of the nature of fresh-cut fruits and vegetables as they relate to pre-and post-harvest handling, processing, packaging and storage are essential for ensur-ing their wholesomeness and nutritional value, and for developing the most effec-tive procedures and innovative technologies for maintaining their quality to meetincreasing consumer demand. Attention to the market and economic factors willalso ensure the ability of the industry to consistently deliver value to consumers,develop and implement new technologies and reward all participants in the supplychain.

This book is a comprehensive interdisciplinary reference source for the emergingfresh-cut fruits and vegetable industry. It focuses on the unique biochemical, phys-iological, microbiological, and quality changes in fresh-cut processing and storageand on the distinct equipment and packaging requirements, production economicsand marketing considerations for fresh-cut products. Based on the extensive researchin this area during the past 10 years, this reference is the first to cover the completespectrum of science, technology and marketing issues related to this field, includingproduction, processing, physiology, biochemistry, microbiology, safety, engineering,sensory, biotechnology, and economics. It will be particularly useful for seniorundergraduate and graduate students, food scientists, plant physiologists, micro-biologists, chemists, biochemists, chemical engineers, nutritionists, agricultural econ-omists, and molecular biologists.

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I am grateful to each of the authors for their participation, promptness andcooperation as well as many others for their contributions, advice and encouragementin the development of this book.

Olusola Lamikanra

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List of Contributors

Tareq Al-Ati

Department of Food ScienceCornell UniversityStocking HallIthaca, New York

Elizabeth A. Baldwin

Agricultural Research ServiceUnited States Department of AgricultureWinter Haven, Florida

Diane M. Barrett

Department of Food Science and TechnologyCruess HallUniversity of CaliforniaDavis, California

John C. Beaulieu

Southern Regional Research CenterAgricultural Research ServiceUnited States Department of AgricultureNew Orleans, Louisiana

Karen L. Bett

Southern Regional Research CenterAgricultural Research ServiceUnited States Department of AgricultureNew Orleans, Louisiana

Jianchi Chen

Division of Agricultural SciencesFlorida A&M UniversityTallahassee, Florida

Jennifer R. DeEll

Horticulture Research and Development Centre

Agriculture and Agri-Food CanadaQuebec, Canada

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Elisabeth Garcia

Department of Food Science and Technology

University of CaliforniaDavis, California

Edith H. Garrett

International Fresh-cut Produce Association

Alexandria, Virginia

Gillian M. Heard

Department of Food Science and Technology

The University of New South WalesSydney, Australia

Joseph H. Hotchkiss

Department of Food ScienceCornell UniversityIthaca, New York

William C. Hurst

Department of Food ScienceUniversity of GeorgiaAthens, Georgia

Jennylynd A. James

Dole Food Company, Inc.Westlake Village, California

Adel A. Kader

Department of PomologyUniversity of CaliforniaDavis, California

Olusola Lamikanra

Southern Regional Research CenterAgricultural Research ServiceU.S. Department of AgricultureNew Orleans, Louisiana

Jérôme Mazollier

Centre Technique Interprofessionel des Fruits et Legumes

France

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Greg Pompelli

Agricultural and Trade Outlook BranchEconomic Research ServiceU.S. Department of AgricultureWashington, D.C.

Peter M. A. Toivonen

Pacific Agri-Food Research CentreAgriculture and Agri-Food CanadaSummerland, British ColumbiaCanada

Patrick Varoquaux

Ministere De L’AgricultureINRAStation de Technologie

des Produits Végétaux France

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Table of Contents

Chapter 1

Fresh-cut Produce: Tracks and Trends

Edith H. Garrett

Chapter 2

Quality Parameters of Fresh-cut Fruit and Vegetable Products

Adel A. Kader

Chapter 3

Overview of the European Fresh-cut Produce Industry

Patrick Varoquaux and Jérôme Mazollier

Chapter 4

Safety Aspects of Fresh-cut Fruits and Vegetables

William C. Hurst

Chapter 5

Physiology of Fresh-cut Fruits and Vegetables

Peter M. A. Toivonen and Jennifer R. DeEll

Chapter 6

Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables

Olusola Lamikanra

Chapter 7

Microbiology of Fresh-cut Produce

Gillian M. Heard

Chapter 8

Microbial Enzymes Associated with Fresh-cut Produce

Jianchi Chen

Chapter 9

Preservative Treatments for Fresh-cut Fruits and Vegetables

Elisabeth Garcia and Diane M. Barrett

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Chapter 10

Application of Packaging and Modified Atmosphere to Fresh-cut Fruits and Vegetables

Tareq Al-Ati and Joseph H. Hotchkiss

Chapter 11

Biotechnology and the Fresh-cut Produce Industry

Jennylynd A. James

Chapter 12

Flavor and Aroma of Fresh-cut Fruits and Vegetables

John C. Beaulieu and Elizabeth A. Baldwin

Chapter 13

Evaluating Sensory Quality of Fresh-cut Fruits and Vegetables

Karen L. Bett

Chapter 14

Future Economic and Marketing Considerations

Greg Pompelli

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Fresh-cut Produce: Tracks and Trends

Edith H. Garrett

CONTENTS

IntroductionSize of the Industry

Fresh-cut ProduceOrganic ProduceImported Produce

Improvements in OperationsImproved Organization of IndustryFoodservice DemandsImprovement of Quality CharacteristicsNew Packaging Technology Shelf Life Improvement

Market PressuresConsolidationLaborCustomer DemandsOther

Food Safety Regulatory Status SummaryReferences

INTRODUCTION

Fresh-cut produce has been one of the hottest commodities in grocery stores overthe past 10 years. The industry soared to over $10 billion in U.S. retail and food-service sales in 1999, and there are no signs of the trend slowing down (IFPA, 2000).In fact, sales for cut and packaged fruit are just getting off the ground, and newcommodities such as cut tomatoes are emerging to answer the consumer’s desire formore convenience in their daily lives.

What is driving this fresh-cut growth? Where did the industry come from, andwhat are the market influences affecting the continued growth of the industry? Wheredoes the processor get ideas for new products, and what track did the processors

1

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take to build success? This chapter will cover the history, current trends and issuesaffecting the fresh-cut produce industry.

SIZE OF THE INDUSTRY

According to the Produce Marketing Association (PMA), the size of the freshproduce industry was $76 billion in sales for 1999, including foodservice and retailsales (PMA, 2000; Kaufman et al., 2000). Fresh produce has always been popularwith consumers because of the wonderful flavors, the natural nutritious quality andfreshness. In fact, the United States Department of Agriculture (USDA) reports thatproduce consumption in the U.S. rose from 284 pounds per capita in 1990 to 319pounds per capita in 1998 (Kaufman et al., 2000).

F

RESH

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RODUCE

All these same attributes, along with added convenience, continue to drive sales forunique fresh-cut commodities. The International Fresh-cut Produce Association (IFPA)defines fresh-cut produce as “any fruit or vegetable or combination thereof that hasbeen physically altered from its original form, but remains in a fresh state” (IFPAand PMA, 1999, p. 5).

IFPA estimates the U.S. fresh-cut produce market at approximately $10–12 billionin sales in 2000, with foodservice sales making up about 60% of the total (IFPA,2000). Packaged salads have been rising stars in the grocery store for the past decade,and, with cut fruits and vegetables included, this category is estimated by IFPA tocontinue to grow in sales in the U.S. retail market at 10–15% a year for the next fiveyears. The category in U.S. foodservice sales is difficult to measure but is estimatedby IFPA to grow 3–5% a year for the next five years.

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RGANIC

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RODUCE

Organically grown fruits and vegetables are another segment of the fresh produceindustry that have experienced strong growth in the 1990s. This category includesboth whole commodities and fresh-cut products. Making up an estimated $4 billionin sales in 2000 (PMA, 2000), the organic produce industry is projected to have anincrease of 7% annually in sales in the next three years. Again, the consumer islooking for healthy, flavorful alternatives for their diets, and organic fresh-cut pro-duce meets these criteria. As the availability of organic produce increases, productioncosts are reduced, making this an affordable product to serve in restaurants and sellin conventional grocery stores. Fresh-cut organic salads are now readily availablein the marketplace.

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MPORTED

P

RODUCE

Consumption of imported commodities has grown in the past decade, and consumersnow enjoy year-round availability of many produce items in the U.S. and Europe.Importation is necessitated by the fact that fruits and vegetables are not grown inany one locale every month. The market for imported produce continues to grow in

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Fresh-cut Produce: Tracks and Trends

3

many parts of the world. The latest USDA reports show that U.S. imports of freshfruits and vegetables accounted for $4.1 billion in sales in 1997, a 105% increaseover 1987’s total of $2 billion (Kaufman et al., 2000).

IMPROVEMENTS IN OPERATIONS

Since the 1940s, produce companies have devised unique ways to cut and packageproduce for sale. Initially, some used bathtubs to wash produce, while others usedthe spin dry cycle on washing machines for the drying step. Ice was used in waterbaths to chill produce, and rudimentary packaging provided little more than protec-tion from contamination during distribution. The industry built much of their ownequipment as production increased in the 1970s from the growth in foodservicesales, but real innovation coincided with an increase in the number of restaurants inthe 1980s.

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MPROVED

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RGANIZATION

OF

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NDUSTRY

Many technological advances occurred in the 1980s and 1990s as the industry becameorganized via their own trade association, the IFPA. Suppliers joined the trade asso-ciation and participated in a growing annual equipment trade show to sell equipmentand network with processors. This new forum for technology exchange helped propelthe industry forward and enhance the quality and safety of fresh-cut produce.

Industry research revealed many new steps for shelf life improvement and con-vinced the industry to focus on refrigeration as the most critical step in the productionprocess. The mantra became “the earlier the chilling step, the better the finishedproduct.” In other developments, major equipment innovations that improved fresh-cut production standards included the closed flume water bath, advanced cutters fora variety of cut sizes, advanced drying machines, the automatic packaging machine,automatic sanitation equipment and electronic monitoring equipment.

Each technological advancement increased production speed but caused new bot-tlenecks. Thus, there has been increased movement toward greater automation andelectronic control by the industry. Today, the design of fresh-cut operations centerson food safety and sanitation, excellent refrigeration, higher production speeds throughautomation, quality enhancement and product traceability.

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OODSERVICE

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EMANDS

In the mid 1970s, restaurants saw a great opportunity to save on labor costs by switch-ing to convenient fresh-cut produce. Meeting the growing demands of McDonald’sand other fast-food chains, growers and processors built the shredded lettuce andchopped onion business into a formidable niche within the fresh produce industry(Lawn and Krummert, 1995).

In the mid 1980s, there was tremendous growth in restaurants in North America.Salad bars became the latest craze with consumers. Soon, fresh fruits and vegetablestook the place of canned produce on salad bars across America. Consistently anindustry innovator, McDonald’s Corporation decided it wanted to eliminate salad

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bars in its stores to reduce food safety risks to consumers. The company asked itssuppliers for a fresh salad to be made and packed in 5-lb. bags that would be repackagedin single-serve trays for sale within its stores.

Mixing commodities together under hermetically sealed packaging was not acommon practice at the time, but the success of the McDonald’s salad motivatedother restaurant chains to provide similar products. This was also a time when womenbegan working outside the home in large numbers, and two-income families feelinga time crunch began looking for more convenience in their lives. Cut and packagedproduce fit those needs perfectly, but the fresh-cut industry at that time could notprovide consistent quality and sufficient shelf life for the retail marketplace. How-ever, these obstacles were soon to be overcome.

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UALITY

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HARACTERISTICS

Even though fresh-cut produce had been sold at retail since the 1940s, it was notcompletely successful, because the quality was unpredictable and the shelf life limited.Initially, processors used cast-offs, blemished product or second-quality commoditiesfor the cut produce. In addition, refrigeration was poor throughout distribution, andappropriate packaging had not been developed. As the demand for better productswith longer shelf life grew from foodservice customers, the industry’s efforts wereconcentrated on quality improvements.

One thing the processors knew — their leading challenge was to stop the producefrom turning brown after it was cut. Product appearance was the primary focus forquality measurement at the time, and processors found that refrigeration alone wasnot going to control discoloration and other visible defects. Instead, they had to startwith healthier raw products, gentler handling procedures during processing and betterpackaging. Today, processors are concentrating on the importance of enhanced flavordevelopment to provide even better ready-to-eat products.

Growers began supplying first quality commodities for processors, and new equip-ment processes were introduced such as air drying and gentle water baths. Someprocessors experimented with chemical washes or edible films to prevent browning,but low rates of improvement did not justify the additional costs. Improved packagingbecame the next step in the quest to address these quality challenges.

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ACKAGING

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ECHNOLOGY

In the 1940s, during the early days of fresh-cut produce, packaging consisted ofcellophane wrappers over cardboard trays for products like coleslaw or salads (Hold-erfield, 1946). Cellophane, styrene and other plastics were used to wrap cauliflowerheads in the mid 1950s in California produce fields to reduce shipping weights andprolong shelf life. In the early 1960s, lettuce growers began wrapping head lettuce.Both products are still popular in today’s retail markets (Anderson, 2000).

The next step for lettuce growers was to trim and core the iceberg heads beforepacking them in plastic bags for shipment to the East Coast. This practice is stillcarried out today, and growers are even packing cleaned and cored lettuce in largebins for shipment to processors around the country.

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In the mid 1980s, the fresh-cut industry was small and fragmented in the U.S.,and packaging suppliers did not focus research efforts on developing films specifi-cally for use with cut produce. European companies, however, were consolidatingand developing equipment and packaging systems to move their industry forward.

New packaging was not as easy to find in the U.S. in the 1980s, because poly-ethylene film was the only breathable film on the market that could preserve produceand hold up to the rough handling conditions. Initially, processors used bags that weredesigned for other foods such as turkey and other meats. The advent of automaticpackaging machines in the late 1980s spurned the development of new and innovativepackaging that solved quality problems and helped launch fresh-cuts into mainstreammarketing and distribution channels.

With the advent of automated packaging machines for fresh-cut produce in thelate 1980s, the plastics industry jumped into action to design materials for fresh-cutproduce. Film companies looked for new polymers and manufacturing processes tocreate breathable films that could run on the automatic machinery. Companies likeMobile, Exxon and Amoco provided new polymers from petroleum products andentered the market to better understand the needs of the industry. Automatic machinesand these new films combined to allow processors to launch smaller, branded bagsfor the new fresh-cut products in the early 1990s.

In 1995, the Flexible Packaging Association (FPA) reported in their annualsurvey of packaging converters that for the first time, produce had overtaken medicalpackaging as the number one product for their production facilities (FPA, 1995).Estimated at $90 million in U.S. sales (Packaging Strategies, 1999), packaging forproduce would be the number one product for the next five years, respondents reportedin the 2000 survey (FPA, 2000).

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MPROVEMENT

Beyond the revolutionary impact on the plastics industry, the processors have alsoinfluenced fruit and vegetable growers to focus on the burgeoning fresh-cut market.Instead of second quality, misshapen commodities or blemished fruits and vegeta-bles, processors ask for first quality and negotiate contracts for the best quality rawproducts they can procure. Today’s trends include growers competing for processorcontracts by committing whole fields to processors, seed companies developing newvarieties to suit the needs of processors and equipment suppliers engineering inno-vative tools to reduce harvesting damage to the produce.

Other engineering feats positively impacting the fresh-cut industry today includeadvanced air-drying techniques to reduce damage to the cut produce, vastly improvedrefrigeration in the processing plants, retail outlets’ increased attention to refrigerationand sanitation and application of HACCP and other food safety systems. Clearly,the industry’s commitment to develop researchers and supplier partners who collab-orate to solve quality and shelf life challenges has resulted in better quality, longershelf life and steady sales growth today.

Today, salads and most vegetables have a 12–14 day shelf life, while fruits are moreperishable and have a shorter shelf life of 8–10 days if held at temperatures between33

°

F (1

°

C) and 41

°

F (5°C) (IFPA and PMA, 1999). Consumers now enjoy fresh-cut

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salads, fruits and vegetables on a year-round basis, and the industry is committed todeveloping better products to continue delivering reliable quality for their customers.

MARKET PRESSURES

In North America, the fresh-cut business is comprised of two general categories ofprocessors. National companies are represented by large grower/shipper/processoroperations, frequently including multiple processing plants in several regional loca-tions, with a main office located in California’s agricultural areas. These grower-based companies are able to focus on a specific commodity such as baby carrots,packaged salads, broccoli or onions. Their facilities are designed for efficiency inthe production of large quantities of a few commodities, and they specialize in sellingto retail and/or foodservice chains.

A second category is made of medium- to small-sized regional processors thatgrew out of produce distribution companies in metropolitan areas. These companiesare frequently family-owned single-facility operations that have evolved in a regionalmarket and are usually designed for flexibility to serve the needs of retail or food-service distributors. Their customer base may order small amounts of a variety ofcommodities to sell to many grocery or restaurant outlets within a defined region,or they may be large distributors for chains that are buying from several regionalfresh-cut operators in different parts of the country. These processors often operateshort production runs of numerous products during the course of a day.

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ONSOLIDATION

The fresh-cut industry has not escaped the influence of recent corporate consolidationtrends. Foodservice and retail buyers are combining at a rapid rate around the world,forcing processors to consolidate (Kaufman et al., 2000). Bigger companies wantto buy from bigger suppliers, and this trend pushes down to the basic level of growersand other suppliers. This domino effect is resulting in the creation of larger proces-sors who sell specific commodity lines to large customers, thus forming partnershipsthat make for tough competition. National operators who are looking for distributionrights, regional locations and volume consolidation are buying regional operations.In some cases, regional companies are combining to form larger companies to supplythe growing foodservice chains.

Nelson (1999) identified 10 innovative options that processors are taking toremain competitive in the consolidating marketplace:

1. Joining the trend and selling out to a larger corporation2. Concentrating on one commodity such as carrots or onions and becoming

specialized in all aspects of that commodity, from growing through brandmarketing (for example, Grimmway Farms’ baby carrots)

3. Forming a strategic alliance with a larger company to process a brandedproduct (for example, Verdelli Farms processing Mann broccoli)

4. Creating a cooperative buying or marketing group to reap the savingsrealized by other larger corporations

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Fresh-cut Produce: Tracks and Trends

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5. Specializing in processing under a private label for store-branded foods6. Co-branding with a non-produce company that wants to have its brand

associated with the successful fresh-cut product line (for example, WeightWatcher’s salads)

7. Choosing a marketing niche for product line focus (for example, organicproduce)

8. Developing or utilizing proprietary technology to set their products apartfrom others

9. Creating new market segments (for example, sliced tomatoes)10. Specializing in the difficult or unusual (for example, hand-carved vege-

tables for luxury hotels and restaurants)

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ABOR

Another pressure felt universally by the fresh-cut industry is a general labor shortage.Company owners continue to plan strategies to find new sources of reliable hourlylabor, but they are rapidly investing their resources toward automation to reducetheir reliance on hourly employees. In developed economies, immigrants make upthe vast majority of the manual labor needed in fresh-cut operations. If immigrationis impeded for any reason, the shortage increases. In addition, a variety of languagesand cultures in one operation can result in barriers to effective training. Theselimitations continue to especially plague smaller operators in the metropolitan areas.

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Aside from the enormous upheaval in the wake of customer consolidation, the fresh-cut industry continues to be influenced by the distribution characteristics, productdevelopment demands and purchasing specifications set by retail and foodservicecorporations. These customers demand that their suppliers drive costs out of thesystem by requiring the use of internet technology for electronic data transfer andcommunication, productivity improvements, food safety audits, approved supplierprograms and other system-wide streamlining.

The safety of produce continues to capture the attention of purchasing agentsin the foodservice and retail sectors. The latest trend in North America is towardrequirements from retailers for third-party food safety audits of growers (Hilton,1999; Wright, 1999). Fresh-cut processors have complied with these types of auditsfor many years from foodservice customers, but this is new for fruit and vegetablegrowers.

As consolidation blurs the boundaries of foodservice and retail companies,exemplified by the recent purchase of PYA/Monarch, a large U.S. foodservicedistributor, by Ahold, the sixth largest global retailer (Reuters, 2000), food safetyand other standards may also blur between the two industries. A retail industrybellwether to watch in the consolidation game is the discount retailer, Wal-Mart, asthey continue to set new standards. Global food chains and their suppliers struggleto keep up with formidable competitors like Ahold and Wal-Mart.

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O

THER

Internet technology growth and increasing government regulation round out the listof major pressures for fresh-cut manufacturers around the world. Food safety regu-lation has been impacting the food industry around the world for the past five yearsand promises to continue to remain in the spotlight. Perhaps one consolation in today’sglobal market is that many countries are working together to create food safetystandards that will affect this industry on an even and fair basis. With food impor-tation and exportation on the rise, it makes sense that new regulations should beharmonized around the world to level the playing field within the global marketplace.

FOOD SAFETY REGULATORY STATUS

The risk of developing foodborne illness from fresh produce is not precisely knownat this time, because the outbreaks associated with fruits and vegetables have beensporadic and incompletely reported. There is even some debate of whether theincidence of foodborne illness associated with produce is on the rise or only trackedand reported more efficiently (Harris et al., 2000). Also, there are no definitiveintervention strategies that assure the elimination of pathogens from fresh produce.Therefore, the industry must focus on the prevention of contamination of freshproduce with human pathogens to assure that these products are safe and wholesomefor human consumption (Gorny and Zagory, 2002).

In the past five years, media stories featuring produce have not been very positive,and the result of this negative attention has been increased regulatory oversight of theproduce industry. In the U.S. and Canada, guidance or regulations have been devel-oped for the safe and hygienic production, harvesting, packing, processing and trans-porting of produce.

Likewise, in Europe, Australia and other countries, new standards or regulationsare addressing contamination issues linked to produce. The international standards-forming body, Codex Alimentarius, hopes to have a document for hygienic proce-dures in the harvesting and packing of fresh fruits and vegetables ready in the nextseveral years. There are currently two annexes to this draft standard, one coveringsprouts and one covering fresh-cut produce (Codex, 2000). This particular initiativewill apply to all countries in the World Health Organization and the Food & Agri-culture Organization to further harmonize the global marketplace.

The food industry has received broad coverage in the news in the last five yearsdue to many issues such as biotechnology, foodborne illness outbreaks and productrecalls. But, according to the International Food Information Council Foundation(IFCF), the tide may be changing to a more positive image for food, and producein particular, in the media.

IFCF reports that the number of food news stories increased from 810 to 1260in May–July 1999, a 38% rise as compared to the same time frame in 1998. Twenty-nine percent of all the coverage measured focused on general wellness and health-boosting aspects of food, and these benefits outweighed negatives 57% vs. 43%.The previous year, the negatives outweighed the benefits, 54% vs. 45%. They also

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Fresh-cut Produce: Tracks and Trends

9

noted that scientific researchers and experts were the most frequently quoted sourcesin food news reporting, which adds credibility to the stories (IFIC, 2000).

Food safety issues are very important, and the industry needs to institute updatedsanitation practices, but the produce industry has a very positive message for theconsumer, because most fruits and vegetables are low in fat and high in fiber andnutrients. A balanced, science-based approach is appropriate for media coverage ofproduce.

SUMMARY

The value of fresh-cut produce lies in the primary characteristics of freshness andconvenience. Food safety, nutrition and sensory quality are required while providingextended shelf life and freshness. Fresh-cut produce is a safe, wholesome food whenproduced under Good Agricultural Practices (GAPs), Good Manufacturing Practices(GMPs) and sanitation procedures. Today’s food marketplace is alive with new prod-ucts and changing trends, and fresh-cut produce remains at the top of the list ofproducts meeting the needs of today’s busy consumers. This publication is providingthe industry an up-to-date summary of the current science and marketing trends toassure that we continue to earn the trust and confidence of consumers everywhere.

REFERENCES

Anderson, B. 2000. “A History of the Packing Industry on the Central Coast,”

Coastal Grower,

Summer Issue: 18–23.Codex Committee on Food Hygiene. 2000. Meeting report from the 32nd session, Washington,

D.C, November 29–December 4, 2000. www.fao.org/waicent/faoinfo/economic/esn/codex.

Flexible Packaging Association (FPA). 2000. “State of the Industry Report.” Business andEconomic Research Division of the FPA, Washington, D.C.

Flexible Packaging Association (FPA). 1995. “State of the Industry Report.” Business andEconomic Research Division of the FPA, Washington, D.C.

Gorny, J.R. and Zagory, D. 2002. “Produce Food Safety” in

The Commercial Storage of Fruits,Vegetables, and Florist and Nursery Stocks,

K.C. Gross, M.E. Saltveit, and C.Y. Wang(eds.), U.S. Department of Agriculture, Agriculture Handbook 66, Washington, D.C.

Harris, L.J., Zagory, D., and Gorny, J.R. 2000. “Safety Factors,” in

Postharvest Technologyof Horticultural Crops,

A. Kader (ed.), Oakland, CA, University of California, Divi-sion of Agriculture and Natural Resources, Special Publication 3311.

Hilton, S.H. 1999. Corporate correspondence to U.S. produce suppliers. Albertsons, Inc.,Boise, ID.

Holderfield, J.W. 1946. “Farmer Brown Builds Big Business by Pre-packaging Produce forRetailers” in

Voluntary and Cooperative Groups Magazine

. February, 8–9, 44, 55–56.International Food Information Council (IFIC). 2000. “Food For Thought III: Reporting of

Diet, Nutrition and Food Safety.” Research report, Washington, D.C. International Fresh-cut Produce Association (IFPA). 2000. “Fresh-cut Produce: Get the Facts!”

Fact sheet published by the association on their web site www.fresh-cuts.org. Alexandria,VA.

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

International Fresh-cut Produce Association (IFPA) and the Produce Marketing Association(PMA). 1999. “Handling Guidelines for the Fresh-cut Produce Industry,” 3rd edition,pp. 5, 7. IFPA, Alexandria, VA.

Kaufman, P.R., Handy, C.R., McLaughlin, E. W., Park, K., and Green, G.M. 2000. “Under-standing the Dynamics of Produce Markets: Consumption and Consolidation Grow.”Food and Rural Economics Division report, Economic Research Service, U.S. Depart-ment of Agriculture. Agriculture Bulletin No. 758.

Lawn, J. and Krummert, B. 1995. “Rise of Fresh-cut: Conquering New Frontiers” in

TheFoodservice Distributor

. August, 46–50, 70.Nelson, C. 1999. “Industry Consolidation: A Survival Course.” IFPA 12th Annual Conference

and Exhibition. April 15–17, 1999. Tampa, FL.Packaging Strategies. 1999. “Technology Expands Along with Fresh-cut Produce Market,”

Packaging Strategies Newsletter,

Westchester, PA.Produce Marketing Association (PMA). 2000. “Retail Fresh Produce Industry Sales” and

“Industry Overview: Foodservice.” Newark, DE.Reuters. 2000. “Sara Lee Completes PYA/Monarch Sale to Ahold.” Article from the Reuters

News Service, December 4, 2000.Wright, E. 1999. Corporate correspondence to U.S. produce suppliers. Safeway, Inc., Walnut

Creek, CA.

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Quality Parameters of Fresh-cut Fruit and Vegetable Products

Adel A. Kader

CONTENTS

Quality ParametersAppearance (Visual) Quality FactorsTextural (Feel) Quality FactorsFlavor (Eating) Quality FactorsNutritional Quality Factors

Preharvest Factors Influencing QualityGenotypes and RootstocksClimatic FactorsCultural Practices

Maturity and RipeningMaturityRipening

Postharvest Factors Influencing QualityPhysical Damage During Harvesting and HandlingTemperature and Relative Humidity ManagementSupplemental Treatments Applied to the CommoditySupplemental Treatments Involving Manipulation of the EnvironmentFlavor vs. Appearance Life of Fresh-cut Fruit Products

Quality Assurance ProgramsReferences

Quality of fresh-cut fruit and vegetable products is a combination of attributes, prop-erties, or characteristics that determine their value to the consumer. Quality parametersinclude appearance, texture, flavor, and nutritive value. The relative importance of eachquality parameter depends upon the commodity or the product and whether it is eatenfresh (with or without flavor modifiers, such as dressings and dips) or cooked. Con-sumers judge quality of fresh-cut fruits and vegetables on the basis of appearance and

2

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freshness (“best if used by” date) at the time of purchase. However, subsequent pur-chases depend upon the consumer’s satisfaction in terms of textural and flavor (eating)quality of the product. Consumers are also interested in the nutritional quality and safetyof fresh-cut products.

Quality of the intact fruit or vegetable depends upon the cultivar, preharvest culturalpractices and climatic conditions, maturity at harvest, and harvesting method. Handlingprocedures, conditions, and time between harvest and preparation as a fresh-cut productalso have major impacts on quality of intact fruits and vegetables and, consequently,quality of the fresh-cut products. Additional factors that influence quality of fresh-cutfruits and vegetables include method of preparation (sharpness of the cutting tools,size and surface area of the cut pieces, washing, and removal of surface moisture)and subsequent handling conditions (packaging, speed of cooling, maintaining opti-mum ranges of temperature and relative humidity, expedited marketing, and propersanitation procedures). An effective quality assurance program must take into con-sideration all the factors that affect quality of the intact fruits or vegetables and theirfresh-cut products.

QUALITY PARAMETERS

A

PPEARANCE

(V

ISUAL

) Q

UALITY

F

ACTORS

These may include size, shape, color, gloss, and freedom from defects and decay.Defects can originate before harvest as a result of damage by insects, diseases, birds,and hail; chemical injuries; and various blemishes (such as scars, scabs, russeting,rind staining). Postharvest defects may be morphological, physical, physiological,or pathological. Morphological defects include sprouting of potatoes, onions, andgarlic; rooting of onions; elongation and curvature of asparagus; seed germinationinside fruits such as lemons, tomatoes, and peppers; presence of seed stems in cabbageand lettuce; doubles in cherries; and floret opening in broccoli. Physical defectsinclude shriveling and wilting of all commodities; internal drying of some fruits;mechanical damage such as punctures, cuts and deep scratches, splits and crushing,skin abrasions and scuffing, deformation (compression), and bruising; and growthcracks (radial, concentric). Temperature-related disorders (freezing, chilling, sunburn,sunscald), puffiness of tomatoes, blossom-end rot tomatoes, tipburn of lettuce, internalbreakdown of stone fruits, water core of apples, and black heart of potatoes areexamples of physiological defects.

Examples of defects that do not influence postharvest life potential of fresh produceinclude healed frost damage, scars, and scabs; well-healed insect stings; irregularshape; and suboptimal color uniformity and intensity. Most other defects (listed above)reduce postharvest life potential of fresh fruits and vegetables.

Tissue browning, which can be a major defect of fresh-cut fruits and vegetables,depends upon the concentration of phenolic compounds, the activity of polyphenoloxidase (PPO), and the concentration of antioxidants in the tissue. Wound-inducedloss of cellular compartmentation between the phenolic compounds (mainly in thevacuole) and PPO (in the cytoplasm) results in tissue browning at a rate that increaseswith temperature and water stress.

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Quality Parameters of Fresh-cut Fruit and Vegetable Products

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T

EXTURAL

(F

EEL

) Q

UALITY

F

ACTORS

These include firmness, crispness, juiciness, mealiness, and toughness depending on thecommodity. Textural quality of fruits and vegetables is not only important for their eatingand cooking quality but also for their shipping ability. Soft fruits cannot be shipped longdistances without extensive losses due to physical injuries. This has necessitated harvest-ing fruits at less than ideal maturity from the flavor quality standpoint in many cases,such as the melons sold during the winter months in the U.S. markets.

Tissue softening and associated loss of integrity and leakage of juice from somefresh-cut products can be the primary cause of poor quality and unmarketability.Increasing calcium concentration in the tissue can slow down its softening rate. Also,initial firmness, temperature, and vibration influence the rate of softening and juiceleakage from fresh-cut fruits.

F

LAVOR

(E

ATING

) Q

UALITY

F

ACTORS

These include sweetness, sourness (acidity), astringency, bitterness, aroma, and off-flavors. Flavor quality involves perception of the tastes and aromas of many com-pounds. Objective analytical determination of critical components must be coupledwith subjective evaluations by a taste panel to yield useful and meaningful informationabout flavor quality of fresh fruits and vegetables. This approach can be used to definea minimum level of acceptability. To find out consumer preferences of flavor of a givencommodity, large-scale testing by a representative sample of the consumers is required.

Flavor quality of most fruits is influenced by their contents of sugars (sweetness),organic acids (acidity), phenolic compounds (astringency), and odor-active volatiles(aroma). More information is needed about the optimum concentration ranges ofthese constituents to assure good overall flavor (based on sensory evaluation) of eachkind of fruit (to satisfy the majority of consumers). Also, future research and devel-opment efforts on objective quality evaluation methods must include nondestructivesegregation of fruits on the basis of their contents of sugars, acids, phenolics, andor odor-active volatiles. In many cases, consumers are willing to pay a higher pricefor fruits with good flavor, and there is a growing trend of high-quality-based storesthat serve this clientele.

N

UTRITIONAL

Q

UALITY

F

ACTORS

Fresh fruits and vegetables play a significant role in human nutrition, especially assources of vitamins (vitamin C, vitamin A, vitamin B

6

, thiamine, niacin), minerals, anddietary fiber. Other constituents that may lower the risk of cancer, heart disease, andother diseases include flavonoids, carotenoids, polyphenols, and other phytonutrients.Postharvest losses in nutritional quality, particularly vitamin C content, can be substan-tial and are enhanced by physical damage, extended storage duration, high temperatures,low relative humidity, and chilling injury of chilling-sensitive commodities.

Nutritional value varies greatly among commodities and cultivars of each com-modity. By using plant breeding and biotechnology approaches, it is possible todevelop genotypes with enhanced nutritional quality and improved flavor quality toencourage consumers to eat more fruits and vegetables (at least five servings per day).

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This can have a major positive impact on human health and should be given highpriority in research and extension programs worldwide.

PREHARVEST FACTORS INFLUENCING QUALITY

G

ENOTYPES

AND

R

OOTSTOCKS

Within each commodity, there is a range of genotypic variation in composition, quality,and postharvest life potential. Plant breeders have been successful in selecting carrotand tomato cultivars with much higher carotenoids and vitamin A content, sweet corncultivars that maintain their sweetness longer after harvest, cantaloupe cultivars withhigher sugar content and firmer flesh, and pineapple cultivars with higher contents ofascorbic acid, carotenoids, and sugars. These are just a few examples of what has beenaccomplished in improving quality of fruits and vegetables by genetic manipulations.However, in some cases, commercial cultivars, selected for their ability to withstandthe rigors of marketing and distribution, tend to lack sufficient quality, particularly flavor.

Rootstocks used in fruit production vary in their water and nutrient uptakeabilities and in resistance to pests and diseases. Thus, rootstocks can influence fruitcomposition and some quality attributes as well as yield, in many cases.

There are many opportunities in using biotechnology to maintain postharvestquality and safety of fresh-cut products. However, the priority goals should be toreduce browning potential and softening rate, to attain and maintain good flavor andnutritional quality to meet consumer demands, and to introduce resistance to phys-iological disorders and/or decay-causing pathogens to reduce the use of chemicals.

A cost/benefit analysis (including consumer acceptance issues) should be usedto determine priorities for genetic improvement programs. For example, increasingthe consumption of certain commodities and/or cultivars that are already high innutritive value may be more effective and less expensive than breeding for highercontents of nutrients.

C

LIMATIC

F

ACTORS

Climatic factors, especially temperature and light intensity, have a strong influenceon composition and nutritional quality of fruits and vegetables. Consequently, thelocation and season in which plants are grown can determine their ascorbic acid,carotene, riboflavin, thiamine, and flavonoids content. In general, the lower the lightintensity, the lower the ascorbic acid content of plant tissues. Temperature influencesuptake and metabolism of mineral nutrients by plants because transpiration increaseswith higher temperatures. Rainfall affects the water supply to the plant, which mayinfluence composition of the harvested plant part and its susceptibility to mechanicaldamage during subsequent harvesting and handling operations.

C

ULTURAL

P

RACTICES

Soil type, the rootstock used for fruit trees, mulching, irrigation, and fertilizationinfluence the water and nutrient supply to the plant, which can affect the nutritionalquality of the harvested plant part. The effect of fertilizers on the vitamin content of

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Quality Parameters of Fresh-cut Fruit and Vegetable Products

15

plants is less important than the effects of genotype and climatic conditions, but theirinfluence on mineral content is more significant. For example, sulfur and seleniumuptake influence the concentrations of organosulfur compounds in

Allium

and

Brassica

species. High calcium content in fruits has been related to longer postharvest life asa result of reduced rates of respiration and ethylene production, delayed ripening,increased firmness, and reduced incidence of physiological disorders and decay. Incontrast, high nitrogen content is often associated with shorter postharvest life dueto increased susceptibility to mechanical damage, physiological disorders, and decay.Increasing the nitrogen and/or phosphorus supply to citrus trees results in somewhatlower acidity and ascorbic acid content in citrus fruits, while increased potassiumfertilization increases their acidity and ascorbic acid content.

There are numerous physiological disorders associated with mineral deficiencies.For example, bitter pit of apples; blossom-end rot of tomatoes, peppers, and water-melons; cork spot in apples and pears; and red blotch of lemons are associated withcalcium deficiency in these fruits. Boron deficiency results in corking of apples,apricots, and pears; lumpy rind of citrus fruits; malformation of stone fruits; andcracking of apricots. Poor color of stone fruits may be related to iron and/or zincdeficiencies. Excess sodium and/or chloride (due to salinity) results in reduced fruitsize and higher soluble solids content.

Severe water stress results in increased sunburn of fruits, irregular ripening ofpears, and tough and leathery texture of peaches. Moderate water stress reduces fruitsize and increases contents of soluble solids, acidity, and ascorbic acid. On the otherhand, excess water supply to the plants results in cracking of fruits (such as cherries,prunes, and tomatoes), excessive turgidity leading to increased susceptibility to phys-ical damage, reduced firmness, delayed maturity, and reduced soluble solids content.

Cultural practices such as pruning and thinning determine the crop load and fruitsize, which can influence composition of fruit. The use of pesticides and growthregulators does not directly influence fruit composition but may indirectly affect itdue to delayed or accelerated fruit maturity.

MATURITY AND RIPENING

M

ATURITY

Maturation is the stage of development leading to the attainment of physiologicalor horticultural maturity. Physiological maturity is the stage of development whena plant or plant part will continue ontogeny even if detached. Horticultural maturityis the stage of development when a plant or plant part possesses the prerequisitesfor utilization by consumers for a particular purpose.

Maturity at harvest is the most important factor that determines storage life andfinal fruit quality. Immature fruits are more subject to shriveling and mechanicaldamage and are of inferior quality when ripe. Overripe fruits are likely to becomesoft and mealy with insipid flavor soon after harvest. Any fruit picked either tooearly or too late in its season is more susceptible to physiological disorders and hasa shorter storage life than fruit picked at the proper maturity.

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All fruits and mature-fruit vegetables, with a few exceptions (such as Europeanpears, avocados, and bananas), reach their best eating quality when allowed to ripenon the tree or plant. However, some fruits are usually picked mature but unripe sothat they can withstand the postharvest handling system when shipped long distance.Most currently used maturity indices are based on a compromise between those indicesthat would ensure the best eating quality to the consumer and those that provide theneeded flexibility in marketing.

For most non-fruit- and immature-fruit-vegetables (e.g., cucumbers, summersquash, sweet corn, green beans, and sweet peas), the optimum eating quality isreached before full maturity. In these vegetables, the problem frequently is delayedharvest, which results in lower quality at harvest and faster deterioration after harvest.

R

IPENING

Ripening is the composite of the processes that occur from the latter stages of growthand development through the early stages of senescence and that results in charac-teristic aesthetic and/or food quality, as evidenced by changes in composition, color,texture, or other sensory attributes.

Fruits can be divided into two groups: fruits that are not capable of continuingtheir ripening process once removed from the plant and fruits that can be harvestedmature and ripened off the plant. The following are examples from each group:

• Group one includes berries (such as blackberry, raspberry, strawberry), cherry,citrus (grapefruit, lemon, lime, orange, mandarin, and tangerine), grape,lychee, muskmelons, pineapple, pomegranate, tamarillo, and watermelon.

• Group two includes apple, pear, quince, persimmon, apricot, nectarine,peach, plum, kiwifruit, avocado, banana, mango, papaya, cherimoya, sapo-dilla, sapote, guava, passion fruit, and tomato.

Fruits of the first group, with the exception of some types of muskmelons,produce very small quantities of ethylene and do not respond to ethylene treatmentexcept in terms of degreening (removal of chlorophyll); these should be picked whenfully ripe to ensure good flavor quality. Fruits in group two produce much largerquantities of ethylene in association with their ripening, and exposure to ethylenetreatment (100 ppm for 1 to 2 days at 20

°

C) will result in faster and more uniformripening. Once fruits are ripened, they require more careful handling to minimizebruising. Fruits in group two must be ripened, at least partially, before cutting toassure better flavor quality in the fresh-cut products.

POSTHARVEST FACTORS INFLUENCING QUALITY

P

HYSICAL

D

AMAGE

D

URING

H

ARVESTING

AND

H

ANDLING

Harvesting method can determine the extent of variability in maturity and physicalinjuries and, consequently, influence composition and quality of fruits and vegetables.Mechanical injuries (bruising, surface abrasions, cuts, etc.) can accelerate loss of water

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Quality Parameters of Fresh-cut Fruit and Vegetable Products

17

and vitamin C and increase susceptibility to decay-causing pathogens. The incidenceand severity of such injuries are influenced by the method of harvest (hand vs.mechanical) and management of the harvesting and handling operations.

Physical damage before, during, and after cutting is a major contributor to tissuebrowning, juice leakage, and faster deterioration of the fresh-cut products.

T

EMPERATURE

AND

R

ELATIVE

H

UMIDITY

M

ANAGEMENT

Keeping intact and fresh-cut fruits and vegetables within their optimal ranges oftemperature and relative humidity is the most important factor in maintaining theirquality and minimizing postharvest losses. Above the freezing point (for non-chilling-sensitive commodities) and above the minimum safe temperature (for chilling-sensitive commodities), every 10

°

C increase in temperature accelerates deteriorationand the rate of loss in nutritional quality by two- to threefold. Delays between har-vesting and cooling or processing can result in quantitative losses (due to water lossand decay) and qualitative losses (losses in flavor and nutritional quality). The extentof these losses depends upon the commodity’s condition at harvest and its temper-ature, which can be several degrees higher than ambient temperatures, especiallywhen exposed to direct sunlight.

The distribution chain rarely has the facilities to store each commodity under idealconditions and requires handlers to make compromises as to the choices of temper-ature and relative humidity. These choices can lead to physiological stress and lossof shelf life and quality. The weakest two links in the postharvest handling coldchain of fresh fruits and vegetables are the retail and home handling systems.

S

UPPLEMENTAL

T

REATMENTS

A

PPLIED

TO

THE

C

OMMODITY

These include curing of “root” vegetables, cleaning, sorting to eliminate defects,sorting by maturity/ripeness stage, sizing, waxing, treating with fungicides for decaycontrol, heat treating for decay and/or insect control, fumigating for insect control,irradiating for preventing sprouting or insect disinfestation, and exposing fruits toethylene for faster and more uniform ripening. In most cases, these treatments areuseful in maintaining quality and extending postharvest life of the produce. However,there is a need to determine the maximum storage period that can be used for eachcommodity between harvest and preparation as a fresh-cut product. Generally, thelonger the storage duration of the intact commodity between harvest and cutting,the shorter the post-cutting life of the products.

S

UPPLEMENTAL

T

REATMENTS

I

NVOLVING

M

ANIPULATION

OF

THE

E

NVIRONMENT

Responses to atmospheric modification vary greatly among plant species, organ typeand developmental stage, and duration and temperature of exposure. Maintainingthe optimal ranges of oxygen, carbon dioxide, and ethylene concentrations aroundthe commodity extends its postharvest life by about 50–100% relative to air control.In general, low O

2

atmospheres reduce deterioration and losses of ascorbic acid infresh produce. Elevated CO

2

atmospheres up to 10% also reduce ascorbic acid losses,

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but higher CO

2

concentrations can accelerate these losses. On the other hand, CO

2

-enriched atmospheres can be beneficial in delaying browning and microbial growthon some fresh-cut fruits and vegetables.

Exposure to ethylene can be detrimental to the quality of most vegetables andshould be avoided by separating ethylene-producing commodities from ethylene-sensitive commodities, by using ethylene scrubbers, and/or by introducing fresh,ethylene-free air into storage rooms. Treating the fruits and vegetables or their fresh-cut products with 0.5–1 ppm 1-methylcyclopropene for about six hours protectsthem against ethylene action.

F

LAVOR

VS

. A

PPEARANCE

L

IFE

OF

F

RESH

-

CUT

F

RUIT

P

RODUCTS

Even under optimum preparation and handling conditions, postcutting life based onflavor is shorter than that based on appearance. More research is needed to identify thereasons for the flavor loss and possible treatments to slow it down and to restore theability of the fruit tissue to produce the desirable esters and other aroma compounds.

Use of calcium chloride or calcium lactate in combination with ascorbic acid andcysteine as a processing aid (two-minute dip) has been shown to be effective in firmnessretention and in delaying browning of fresh-cut fruits. Ethylene scrubbing and modifiedatmosphere packaging (to maintain 2–5% O

2

and 8–12% CO

2

) can be useful supple-ments to good temperature management in maintaining quality of fresh-cut fruit prod-ucts. Additional research is needed to optimize preparation and subsequent handlingprocedures for maintaining quality and safety of each fruit product.

QUALITY ASSURANCE PROGRAMS

An effective quality assurance system throughout the handling steps between harvestand retail display is required to provide a consistent good-quality supply of fresh-cut fruits and vegetables to the consumers and to protect the reputation of a givenmarketing label. Quality assurance starts in the field with the selection of the propertime to harvest for maximum quality. Careful harvesting is essential to minimizephysical injuries and maintain quality. Each subsequent step after harvest has thepotential to either maintain or reduce quality; few postharvest procedures can improvethe quality of individual units of the commodity.

Exposure of a commodity to temperatures, relative humidities, and/or concen-trations of oxygen, carbon dioxide, and ethylene outside its optimum ranges willaccelerate loss of all quality attributes. The loss of flavor and nutritional quality offresh intact or cut fruits and vegetables occurs at a faster rate than the loss of texturaland appearance qualities. Thus, quality assurance programs should be based on allquality attributes, not only on appearance factors as is often the case.

Following is a list of handling steps and associated quality assurance functions:

1. Training workers on proper maturity and quality selection, careful handling,and produce protection from sun exposure during harvesting operations

2. Checking product maturity, quality, and temperature upon arrival at theprocessing plant

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Quality Parameters of Fresh-cut Fruit and Vegetable Products

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3. Implementing an effective sanitation program to reduce microbial load4. Checking packaging materials and shipping containers to ensure they meet

specifications5. Training workers on proper processing and packaging operations6. Inspecting a random sample of the packed product to ensure that it meets

grade specification7. Monitoring product temperature to assure completion of the cooling pro-

cess before shipment8. Inspecting all transport vehicles before loading for functionality and clean-

liness9. Training workers on proper loading and placement of temperature-record-

ing devices in each load10. Keeping records of all shipments as part of the “trace-back” system11. Checking product quality upon receipt and moving it quickly to the appro-

priate storage area12. Shipping product from distribution center to retail markets without delay

and on a first-in/first-out basis unless its condition necessitates a differentorder

REFERENCES

Beaudry, R.M. 1999. Effect of O

2

and CO

2

partial pressure on selected phenomena affectingfruit and vegetable quality.

Postharv. Biol. Technol

. 15:293–303.Brecht, J.K. 1995. Physiology of lightly processed fruits and vegetables.

HortScience

.30:18–22.

Ferguson, I., Volz, R., and Woolf, A. 1999. Preharvest factors affecting physiological disordersof fruit.

Postharv. Biol. Technol

. 15:255–262.Goldman, I.L., Kader, A.A., and Heintz, C. 1999. Influence of production, handling, and

storage on phytonutrient content of foods.

Nutrition Reviews.

57(9):S46–S52.Kader, A.A. (ed.). 1992.

Postharvest Technology of Horticultural Crops,

second edition. Publ.3311, Univ. Calif., Div. Agr. Nat. Resources, Oakland, CA, 296 pp.

Kader, A.A. 1999. Fruit maturity, ripening, and quality relationships.

Acta Hort

. 485:203–208.Kays, S.J. 1999. Preharvest factors affecting appearance.

Postharv. Biol. Technol

. 15:233–247.Lee, S.K. and Kader, A.A. 2000. Preharvest and postharvest factors influencing vitamin C

content of horticultural crops.

Postharv. Biol. Technol.

20:207–220.Mattheis, J.P. and Fellman, J.K. 1999. Preharvest factors influencing flavor of fresh fruits and

vegetables.

Postharv. Biol. Technol

. 15:227–232.Paull, R.E. 1999. Effect of temperature and relative humidity on fresh commodity quality.

Postharv. Biol. Technol

. 15:263–277.Romig, W.R. 1995. Selection of cultivars for lightly processed fruits and vegetables.

Hort-Science

. 30:38–40.Saltveit, M.E. 1999. Effect of ethylene on quality of fresh fruits and vegetables.

Postharv.Biol. Technol

. 15:279–292.Sams, C.E. 1999. Preharvest factors affecting postharvest texture.

Posthar. Biol. Technol

.15:249–254.

Shewfelt, R.L. and Brückner, B. (eds.). 2000.

Fruit and Vegetable Quality, An Integrated View

.Technomic Publ. Co., Lancaster, PA, 330 pp.

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Watada, A.E., Ko, N.P., and Minott, D.A. 1996. Factors affecting quality of fresh-cut horti-cultural products.

Postharvest Biol. Technol.

9:115–125.Watada, A.E. and Qi, L. 1999. Quality of fresh-cut produce.

Postharv. Biol. Technol

.15:201–205.

Weston, L.A. and Barth, M.M. 1997. Preharvest factors affecting postharvest quality ofvegetables.

HortScience

. 32:812–816.Wiley, R.C. (ed.). 1994.

Minimally Processed Refrigerated Fruits and Vegetables

. New York:Chapman & Hall, 368 pp.

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Overview of the European Fresh-cut Produce Industry

Patrick Varoquaux and Jérôme Mazollier

CONTENTS

IntroductionHistory of Fresh-cut Fruits and Vegetables in EuropeDevelopment and Statistics

General Processing Conditions Forward-Only MovementSeparation of the Trimming Room, the Washing Room,and the Packing RoomTemperature ControlAirflowWastesCleaning Equipment, Material, and UtensilsSanitationHygienic Procedure for OperatorsChlorinating Distribution Conditions: Chill Chain and Sell-by-Date

Unit OperationsRaw MaterialsHarvestingQuality AssessmentTrimmingSlicing and Shredding .PrewashingWashing with Chlorinated WaterDrainingWeighing and Packing

ConclusionNew Products

Fresh-cut Fruits Vegetable Mixes

3

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Niche ProductsNovel Processing Techniques

Automatic TrimmingChlorine-Free Fresh-cut CommoditiesModified Atmosphere Packaging (MAP) Prevention of Temperature Abuse

References

INTRODUCTION

H

ISTORY

OF

F

RESH

-

CUT

F

RUITS

AND

V

EGETABLES

IN

E

UROPE

When research into optimal processing of fresh-cut produce began in France about20 years ago, the per capita consumption of fruits and vegetables had steadilydeclined since 1971 due to the development of catering and the integration of womenin the task force (Scandella and Leteinturier, 1989). As a consequence, the timedevoted to meal preparation was reduced accordingly. Moreover, fruits and vegeta-bles are short-lived commodities hardly compatible with one shopping trip a week.As shown in Figure 3.1, the reduction in butterhead lettuce consumption exceeded25% from 1971 to 1982. It is noteworthy that easy-to-use vegetables such as tomatoand endive tips (witloof) did not follow this trend.

This trend alarmed nutritionists and supervisors of supermarket fresh fruit andvegetable departments. During a visit to the United States in the 1970s, ClaudeChertier, fruits and vegetables buyer with Monoprix (French supermarket chain),noticed the salad bar in fast-food restaurants and supermarkets and decided to adapt

FIGURE 3.1

Per capita consumption of vegetables in France in 1971 and 1982 (Scandellaand Leteinturier, 1989).

0

1

2

3

4

5

6

7

8

9

Tomato

Butter

lettu

ce

Endive

shoo

t

Carro

t

Green

bean

Caulifl

ower

Leek

Others

Per

cap

ita c

onsu

mpt

ion

of v

eget

able

s (k

g/y)

1971 1982

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23

the concept of “ready-to-eat” salads to the French market. Claude Chertier got intouch with INRA (National Agronomic Research Institute) to develop his idea (1980).

Shredded celeriac and carrot, along with shredded iceberg lettuce, were alreadyavailable in Northern Europe (1970), but these unpacked products (sometimes justoverwrapped with stretchable PVC), mostly designed for catering, were not adaptedto the French market, because their organoleptic and hygienic quality was poor, theirshelf life was limited to two to three days, and iceberg lettuce was not popular inFrance. At this time, some French processors were already manufacturing precutfresh vegetable mixes for soups.

Claude Chertier wanted the new range of products to be recognized as fresh,safe, and user-friendly. The technical specifications were that the salads (200–300grams) should be packed in order to facilitate supermarket distribution and to preventmicrobial cross-contamination, the products should be distributed at room tempera-ture (around 20

°

C), the shelf life should reach seven days plus an additional twodays in the consumer’s possession, the salad composition should be adjusted to thetaste of French consumers, and processing should not include any additives.

The proposed ingredients were broad-leaved endive (

Cichorium intybus

L. cv

latifolia

), curly endive (

Cichorium intybus

L.), red Italian chicories such as variegated-leaved chicory (i.e.,

chioggia

cv), sugar loaf, lamb’s lettuce (

Valerianella locusta

L.), and some lettuces such as romaine and butterlettuce (

Lactuca sativa

L.) for themixed salads. In order to offer consumers an acceptable range of salad, ClaudeChertier also asked for packed shredded carrot (

Daucus carota

L.) and celeriac(

Apium graveolens

L.) plus shredded red and white cabbages (

Brassica oleracea

L.). From 1981 to 1983, INRA therefore studied their first plant model, broad-leaved

endive. The experiments on the effect of unit operations on physiological disorders,bacterial spoilage, and discoloration of the leaves resulted in a realistic process. Obvi-ously, a shelf life of nine days was not attainable at 20

°

C but was possible at 4–6

°

C.In 1983, the procedure for each operation units of processing was established, and twoprocessors invested in rudimentary processing equipment. At this time, the equipmentwas selected from other processing methods such as canning and freezing and wasnot well adapted to the fresh-cut industry. In 1984, a Swiss equipment manufacturerstarted to produce specific machines for the new fresh-cut industry. The production of“ready-to-use” fresh salads in France amounted to only 1400 metric tons in 1984, buttheir success was immediate since the production reached 8000 metric tons in 1985.These new products were rapidly known as “quatrième gamme” or “fourth range” incommercial terminology. Fruits and vegetables are fresh in the first range, canned inthe second, frozen in the third, and fresh-cut or minimally processed in the fourth.

In 1985, CTIFL (Fruit and Vegetable Professional Technical Center) and otherorganizations such as ADRIA (Association for Agro-food Research and Develop-ment) Normandy, Pasteur Institute (Lyon), and different CRITT (Regional Centerfor Technology Transfer) were also involved in the development of the fresh-cutindustry and provided processors with technical assistance. As a consequence, INRAfocused its activity on a more theoretical approach to the field of the physiologyand microbiology of “fresh-cut” plant tissues. Since the new produce was thoughtto be potentially hazardous, INRA undertook extensive research into the microbialhazards associated with prepacked plant tissues.

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At the same time, the fresh-cut industry’s approach spread throughout northernEurope, and a survey (Anonymous, 1986) concerning minimally processed vegeta-bles counted eight processing units in Holland, four in Belgium, 11 in Germany, atleast two large units in England, five in Switzerland (plus numerous small unitsaround the cities), and 19 in France. The concept of ready-to-eat salad was not assuccessful in southern Europe. There, shelf life ranged from four to six days in thechill chain (from 2–4

°

C). At the same time, most European food-processing machin-ery developed specific processing lines fitted with American, Japanese, and Europeanequipment. Bottled gas companies and film manufacturers proposed new gas mix-tures and films designed to optimize actively and passively modified atmospheres.

D

EVELOPMENT

AND

S

TATISTICS

After this development period, around 1990, there were up to 70 producers in France.Most manufacturers operated under poor hygienic conditions, and the chill chainwas not respected either by transporters or by distributors. The visual quality ofmost fresh-cut produce at the end of their shelf life was poor. These factors inhibitedindustry growth (Figure 3.2). Fresh-cut processing was, nevertheless, responsiblefor a dramatic increase in the consumption of lamb’s lettuce that had been steadilydeclining. This salad, which is grown on sandy soil, is difficult to wash. Presently,the production of fresh-cut lettuce is increasing (10–20% a year) in all Europeancountries. In 1999, the annual tonnage production of fresh-cut leaf lettuce was,respectively, 45,000 in the UK, 39,000 in France, 21,000 in Italy, 20,000 in Germany,10,000 in Spain and Netherlands, and 8,000 in Benelux.

In order to stop the decline and restore hygienic processing and distribution,CTIFL and DGCCRF (a French governmental organization similar to the AmericanFDA) published a guideline for the fresh-cut produce industry. This guideline wasturned into a regulation in 1988 (Anonymous, 1988) and was modified in 1993(Anonymous, 1993), and was then modified again in 1996 (Anonymous, 1996). Itsenforcement resulted in a rapid improvement in the quality and in a dramatic decline

FIGURE 3.2

High and low estimates of fresh-cut produce production in France (Sabino, 1990).

0

10,000

20,000

30,000

40,000

50,000

1980 1985 1990 1995 2000

Pro

duct

ion

(met

ric to

ns)

max min

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Overview of the European Fresh-cut Produce Industry

25

in the number of processing companies. In 1998, four companies were responsiblefor 80% of the fresh-cut production. This trend was identical in all European countries.

In spite of an attempt to diversify the range of commodities proposed to theconsumer (more details in the conclusion), fresh-cut green salads still account forabout 85% of the overall production, as they did in 1986 (Figure 3.3).

GENERAL PROCESSING CONDITIONS

Processors apply HACCP principles as described in

Codex Alimentarius

(annex toCAC/RCP 1–1969, Rev. 3–1997) and in the code of hygienic practices for refriger-ated packaged foods with extended shelf life (Alinorm 99/13, pp. 41–57) for allexisting product types and for new product designs.

The guidelines for fresh-cut processing adapted by the French Administrationare aimed at reducing biological, physical, and chemical hazards associated withthis new type of produce. It proposes conditions under which raw materials aregrown, as well as processing and distribution guidelines. In this review, detailsconcerning recommendations and legislation that are specific to fresh-cut processingare presented.

F

ORWARD

-O

NLY

M

OVEMENT

This requires that there should be no “crossing over” in the processing line betweenthe raw material and clean products.

The examples in Figure 3.4 show that the forward-only principle does not imposea linear processing, but it tolerates no crossing over (product line or waste disposal).

FIGURE 3.3

Percent of the different fresh-cut vegetables in 1986 (Scandella and Leteinturier,1989).

0 10 20 30 40 50 60 70 80

Mixed salads

Other salads

Grated carrots

Grated celeriac

Shredded cabbages

Veg. soup

Radishes

Other

% of production in France

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

S

EPARATION

OF

THE

T

RIMMING

R

OOM

,

THE

W

ASHING

R

OOM

,

AND

THE

P

ACKING

R

OOM

In order to prevent cross-contamination, the different processing rooms must bedelimited by walls in order to progressively increase cleanliness from the trimmingroom to the packaging section (Figure 3.5).

T

EMPERATURE

C

ONTROL

Units are designed and equipped in such a way that the temperatures inside thedifferent rooms are in accordance with the requirements summarized in Figure 3.6.According to French regulation, fresh-packed products must be immediately storedat 4

°

C and maintained at 0–4

°

C until delivered to consumers.

FIGURE 3.4

Principle of the forward-only movement.

FIGURE 3.5

Segmentation of the processing line.

Raw material

Clean product

OK

Raw material

Clean product

Not acceptable

TrimmingRoom

WashingDisinfectingRinsing

PackagingCartonExpedition

At least one wall One wall

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Overview of the European Fresh-cut Produce Industry

27

The following are therefore recommended:

• limit exposure to temperatures above 10

°

C• refrigerate the product at 0–2

°

C before packing in order to be at the righttemperature during the operation

• maintain this temperature during storage

The temperature gradient and flow of products run countercurrently. Temperaturein the trimming and disinfecting rooms must not exceed 12

°

C and must not exceed4

°

C in the packing room and warehouse.

A

IRFLOW

Ventilation systems are designed to maintain the required temperature and preventboth condensation and circulation of dust. The air current must flow from the packingto the trimming room (Figure 3.6).

W

ASTES

Waste materials are evacuated from the facility to avoid any cross-contamination(Figure 3.7).

Inside the premises, equipment and machinery used for nonedible material andwaste must be clearly identified and never used for edible products. Moreover, theyshould be easy to wash and sanitize.

FIGURE 3.6

Temperature gradient and airflow in the processing unit.

FIGURE 3.7

Waste disposal.

Airflow Positive pressure

Ambient T 12°C 4°C 4°CWashing

Rawmaterials

TrimmingPrewashing

DisinfectingRinsingDraining

Packing CartonExpedition

Waste evacuation conveyer

Trimming conveyer

WASTE PRODUCTFLOW

TRIMMING ROOM

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Outside the premises, any reusable receptacle for nonedible material and wasteshould be waterproof and easy to wash and sanitize.

C

LEANING

E

QUIPMENT

, M

ATERIAL

,

AND

U

TENSILS

Washing should be performed by any method or combination of methods involvingmechanical action (scrubbing, brushing, water jet spraying) or chemical cleaning(acidic or alkali detergent). The washing must include the removal of objectionablematter of any sort. A detergent or a disinfecting detergent should be applied so asto permit the elimination of dust and bacterial biofilms.

Efficient rinsing with potable water should eliminate the detached particles anddetergent residues.

S

ANITATION

After washing the premises, the machines must be submitted to an efficient disin-fecting, either by using steam or chemicals.

H

YGIENIC

P

ROCEDURE

FOR

O

PERATORS

Personnel should know the hygienic procedure (International Code of Practice,General Principles of Food Hygiene) and wear protective clothing and footwearspecific to the area.

C

HLORINATING

Use of chlorine, associated with hygienic processing, permits a significant improve-ment in the microbiological quality of the product. According to French Regulations,chlorine disinfection must be followed by rinsing with potable water (less than0.5 ppm active chlorine).

There are different forms of chlorine in water solution. A part of dissolved chlorinecombines immediately with organic matters (combined chlorine). The remaining partis the “free” chlorine. Concentration of free chlorine, which averages 80% of totalchlorine, may be assessed using a specific electrode (which also permits automaticregulation of chlorine content) or a spectrophotometric method with DPD (N,N–diethylphenylene–1,4 diamine) as a reagent. Considering the instability of the chlorine solu-tion, frequent determinations are required.

In most disinfecting equipment, there is a very large dispersion in transit timeof the vegetable chunks. The recommended mean duration of disinfection is 2minutes. pH is an important factor for chlorine efficiency. The pH of the disinfectingsolution should range between 6.5–8. Microbial load (aerobic mesophilic bacteria)changes during processing are shown in Figure 3.8.

D

ISTRIBUTION

C

ONDITIONS

: C

HILL

C

HAIN

AND

S

ELL

-

BY

-D

ATE

In order to maintain produce quality until the time of purchase, fresh-cut manufac-turers must stamp the “best before date” on the bag. Determination of the shelf lifeis the processor’s responsibility. The shelf life of the product must be establishedusing scientific data, taking into account the chill chain temperature.

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29

In order to simulate a realistic distribution of fresh-cut commodities, the tem-perature profile is two-thirds of the shelf life duration at prescribed temperature(4

°

C) and the remaining one-third at 8

°

C. The following are the microbial limits forfresh-cut commodities in France (Anonymous, 1993):

Listeria monocytogenes

and

Salmonella

should not be present in the final product (five samples of 25 g), butonly 100 cfu

g

1

Listeria is tolerated at consumption.

Escherichia coli

tests are done to ensure that contamination is within the followinglimits: for five samples of 25 g, no count should exceed 100 cfu

g

1

, and three outof five should be below 10 cfu

⋅g−1. These conditions are similar to those recom-mended by the International Commission on Microbiological Specifications forFoods (ICMSF, 1986, 1988). The Good Manufacturing Practice Guide also recommendsthat aerobic mesophilic flora be lower than 5⋅106 cfu⋅g−1 with three out of five countsbelow 5⋅105 cfu⋅g−1 in bags after processing. This recommendation is not enforceableand cannot be attained for some commodities such as aromatic herbs (parsley, tarragon,chives, sweet basil, and coriander leaves, which are processed in France) and sproutedseeds.

UNIT OPERATIONS

RAW MATERIALS

It is obvious that the quality of the raw material is one of the most essential factorsdetermining the quality of the final product. Green salads should be, as far as possible,cultivated in open fields. Broad-leafed and curly endives must be etiolated in the fieldin order to increase the processing output using either a rubber band or a plastic bell.This operation should be carried out carefully so as to avoid overstressing etiolated planttissues. For hygienic reasons, no manure or fertilizer of animal origin should be used.

FIGURE 3.8 Microbial count (cfu⋅g−1) during fresh-cut processing of green salads (Scandellaand Leteinturier, 1989).

0

1

2

3

4

5

6

7

8

Raw m

ateria

l

Trimming

Cutting

Was

hing

Draini

ng

Packin

g

Bac

teri

al c

ount

s lo

g(cf

ug-1

)

Acceptable Good

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30 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Numerous research projects in many countries, including the United States,Australia, and France are assessing the suitability of salad cultivars for minimallyprocessing from the processor’s point of view.

The main criteria in assessing this suitability of cultivars to fresh-cut processingare as follows:

1. Processing yield—for example, the output of butter lettuce ranges from25–30% and reaches 50% for broad-leaved endive (Scandella and Letein-turier, 1989)

2. Low sensitivity to physiological disorders and microbial diseases3. Mechanical resistance of the tissue4. Resistance to elevated CO2 concentration (Varoquaux et al., 1996) and/or

low oxygen5. High sugar contents because sugar depletion may be responsible for energy

stress (Forney and Austin, 1988)6. Low respiration rate (Varoquaux et al., 1996)7. Special requirements—for example, all leaves of butter lettuces must be

released when coring, because this salad is not cut thereafter in the process(Scandella and Leteinturier, 1989)

HARVESTING

• Most of the raw material for fresh-cut processing is cultivated undercontracts that specify the cultivars and cultivation techniques (includingacreage, sowing time, pesticide and fertilizer applications, and harvestconditions).

• It is required that the salads be harvested in the morning because of thecooler temperature, but the sugar content of the leaves is higher late inthe afternoon.

• It is well known that produce should be precooled to 1°C as soon aspossible after harvesting in order to extend the potential shelf life. Oneof the conditions required for processors to achieve the quality distinctioncalled “Label Rouge” is vacuum cooling of the salads at 1–2°C withinfour hours after harvest.

• Most salads, except lamb’s lettuce, which is more resistant, should beprocessed within two days. Radicchio can be stored for up to two months.

QUALITY ASSESSMENT

The first operation on receipt of the raw materials is quality control, which isnecessary to achieve a standard product quality. The main criteria are the appearanceof the salads, including overall freshness, the absence of insects, physiological andmicrobial diseases, presence of necrotic tissue, and compliance with regulations onpesticide residues and nitrate content. With some salads stored, as variegated-leafedItalian chicory, for example, the absence of pathogenic bacteria such as Listeriamonocytogenes is checked. All quality assessments are noted on an input grid tocomply with “tracing” requirements.

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Overview of the European Fresh-cut Produce Industry 31

TRIMMING

The required proportions of the ingredients in salad mixes are achieved during trim-ming. The trimming table is supplied with the final percentage of each salad, takinginto account their respective processing output. All unwanted parts of the plant,including most of the outer green leaves and core area, are removed manually. Thisoperation causes injury that could be minimized by using very sharp knives (Bolinand Huxsoll, 1991). This is much easier said than done. In fact, classical stainlesssteel used to manufacture blades is rather soft, and intensively used knives should besharpened very often (every hour or so). Carbon steel used for scalpel blades is brittle,may be dangerous for operators, and releases iron ions that may be involved in browndiscoloration. Ceramic blades are also breakable and are very expensive. Trimmingmay be partly mechanized, at least for broad-leaved and curly endives. A new automatictrimmer was developed by a French company (Soleco SA). This patented machine(U.S. patent 5,421,250; 1995) improves the yield of raw material from 5 to 10 pointsand reduces the manpower cost by a factor of two. The use of the trimmer is, however,limited to broad-leafed and curly chicory. Mechanization of the trimming of butter-head lettuce is more complex. Wounding of plant tissue results in leakage of enzymesand their substrates that are normally in different cell compartments. The destructionof cell microstructures leads to biochemical spoilage such as texture breakdown, off-flavor, and browning (Varoquaux and Wiley, 1996).

One of the most conclusive examples of the effect of wounding on firmness wasobserved on kiwifruit after slicing. The slices lose about 50% of their initial firmnesswithin two days at 10°C. It appears as if this phenomenon was due to the releaseof enzymes with pectinolytic and proteolytic activities by injured cells (Varoquauxet al., 1990).

It is well known that bruising or cutting plant tissues with browning capabilitywill result in a brown discoloration. Because most green salads contain polyphe-noloxidases and phenolic substrates, mainly chlorogenic acid, caffeoyl tartaric ester,and caffeoyl shikimic ester (Goupy et al., 1990, 1994), browning of the cut surfaceis a major problem for minimal processing. As previously mentioned, browning canbe reduced by using very sharp blades and chill storage, but another extremelyimportant factor is the interval between slicing and washing. This was demonstratedwith apple slices.

In Figure 3.9 the reflectance absorbance difference at 440 nm of apple slices fortwo cultivars is reported as a function of time after slicing. The slices cut in air weredipped into water 30 seconds after slicing. Browning of these slices appeared as apeak in absorbance in the 400–440 nm region of the spectrum. Surprisingly, theslices cut in water did not visually turn brown for a few hours when stored at 8°Cunder air. Browning did not affect internal tissue, because no discoloration wasobserved when the slices were cut again.

It is most likely that the prevention of browning in slices cut in water is due tothe instant washing out of cell sap liberated by cutting. In slices cut in air and rapidlydipped into water, the exudate immediately diffused into inner tissue layers prior towashing. The longer the interval between cutting and washing, the browner the slicesturned during storage. A similar phenomenon occurs with cut salad leaves.

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32 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

It seems unrealistic to trim the salads manually under water. To reduce discol-oration, new trimming tables were fitted with a hydraulic flow (upper part) to conveythe trimmed parts and a belt conveyor (lower part) to evacuate the wastes, as shownin Figure 3.10.

SLICING AND SHREDDING

Chicory leaves are cut into 2–3 cm pieces using rotating blades (perpendicular tothe flow) or disk knives (parallel to the flow). This process also causes injury toplant tissue that could be minimized by using very sharp blades sharpened once ortwice a day. When trimming, salad leaves must be washed immediately after cutting;

FIGURE 3.9 Absorbance of apple slices cut in air (open symbols) and cut under water (closedsymbols) as a function of time for two cultivars (Kuczinski et al., 1993).

FIGURE 3.10 Trimming table fitted with water conveyor (upper part) to convey the trimmedproduct. (Photo courtesy of Turatti.)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 5 10 15 20 25 30 35

Time after cutting (min)

Ab

sorb

ance

dif

fere

nce

at

440

nm

Delbard Festival Early Redone

air

water

Visible discoloration

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Overview of the European Fresh-cut Produce Industry 33

any delay in prewashing will enhance browning. Washing the leaves after cutting isessential to prevent browning in the same way as trimming. In most processing lines,the product immediately drops into a washing tank after shredding. Since the cuttingshould take place under water, one of the approaches tested in France was water jetcutting (Béguin et al., 1995). Because the internal liquid of injured cells is removedby the water flow, browning is markedly reduced compared to any commercialcutting techniques. The principle of this machine is shown in Figure 3.11. The leaves(3) are conveyed (1) to a multi-U-shaped grooved belt (2) designed to position themain nervure of the leaves parallel to the direction of flow and to limit the thicknessof the products to two or three layers on the stainless steel grill conveyor (4). Theleaves (or other plant tissues) are cut by the transversal and alternative displacement ofa water jet (6) on a fixed rail (5). The water jet pressure ranges between 50–100 MPadepending on the product to be sliced. The average width (cm) of the chunks is P/2v,where P is the period of the water jet cross-head (min−1), and v is the conveyor velocityin cm⋅min−1 (7). The cut products are dropped into the washing (8).

PREWASHING

When the salad leaves are cut, they fall into the prewasher that washes away exudatesand saps that would otherwise rapidly pollute the disinfecting tank.

WASHING WITH CHLORINATED WATER

The maximum active chlorine allowable in the disinfecting tank was set at 120 ppmby law in France in 1988, and the 1992 guidelines, which are still valid, proposedreducing it to 80 ppm. In current processing, the minimum chlorine concentrationshould not drop below 50 ppm. Chlorine may be hypochlorite or chlorine gas. Thelatter is more complex to handle but is slightly more efficient due to a noticeable decreasein pH of the disinfecting solution. Conversely, addition of hypochlorite increasesthe pH, resulting in a bigger dissociation of hypochlorite and, thus, in a decrease indisinfecting efficiency. In some processing units, the chlorine concentration is mon-itored and adjusted continuously, while in others, it is measured and readjusted everyhour or so. Agitation in the disinfecting tank is insured either by tangential airbubbling or water jets or mechanically by rotating arms (Figure 3.12). It should be

FIGURE 3.11 Sketch of a water jet cutter for fresh-cut commodities (Béguin et al., 1995).

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34 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

noted that chlorine is not actually authorized but only tolerated by French regulationsfor disinfecting minimally processed products. Its use is, however, banned in someEuropean countries such as Belgium, Germany, and Holland. The current trend isto eliminate chlorine from the disinfection process. In the washing equipmentdescribed below, a minimal chlorine concentration of 8 ppm was established as anefficient safeguard against possible contamination by pathogenic bacteria.

In previous work, American researchers stated that there was a significant rela-tionship between the initial bacterial load and the spoilage of shredded iceberg lettuce(Bolin et al., 1977). Nguyen-The and Carlin (2000) reported a clear relationshipbetween the number of aerobic mesophilic bacteria at the end of shelf life andspoilage on fresh-cut broad-leaved endive packed in sealed polypropylene film, butmicrobial pollution may be the consequence of the decay as stated by Carlin et al.(1989). Varoquaux and Wiley (1996) claimed that injury stress at processing andphysiological disorders induced by detrimental packaging conditions along withtemperature abuse were the main causes of the premature decay of fresh-cut produce.

The growth rate of aerobic mesophilic bacteria in highly disinfected salad is higherthan that in control samples washed in tap water (less than 0.5 ppm free chlorine).After a 2 log reduction in bacterial count, due to an efficient sanitation, the bacteriapopulation was identical to the untreated sample after only four days at 10°C (Carlinet al., 1996). The growth of Listeria monocytogenes under the same conditions isdramatically enhanced compared to that of the untreated control (Figure 3.13).Elimination of the saprophytic flora may favor the development of unwanted bacteriasuch as Listeria monocytogenes, which grows faster in highly disinfected samples.

It was postulated that a 2 log reduction in microbial count after disinfection wasnot necessary to ensure product quality. That is why alternative milder sanitationprocesses were developed (see Conclusion).

The last step of the washing operation is a rinsing with tap water containing lessthan 0.5 ppm active chlorine. This unit operation is necessary only when chlorineat a concentration higher than 1 ppm is used. The cold water (1–3°C) must becontinuously renewed in order to avoid chlorine accumulation from the disinfectingsection. This rinsing water can be recycled to the upstream washer after filtrationand chlorinating.

FIGURE 3.12 Conventional washer: (1) sediment discharge valves, (2) ventilator with blow-ing system, (3) pumps and hydraulic manifold, (4) level gauges, (5) cold water nozzles, (6)drum outlet filter, (7) insect removal drum, and (8) flow adjusting drum. (Courtesy of Turatti.)

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Overview of the European Fresh-cut Produce Industry 35

The new washer, illustrated in Figure 3.14, is based on the succession of torrentialand laminar flows. The salad is elevated to the feeding hopper (3) filled with chlori-nated water (5–8 ppm of active chlorine). The chlorine concentration is regulated witha specific electrode, and the recycled solution is filtered. The product is swept alongin the overflowing water (11). The bottom of the first section (1) is equipped withbumps (10) which, combined with the optimal slope α of the disinfecting section, resultsin a succession of laminar and torrential flows. The surface of the commodity is washedin a permanently renewed turbulent chlorine solution. This process also eliminatesaphids. At the end of the first section, the product is separated from the chlorinesolution onto a perforated conveyor (5). The water is returned (7) to the water buffertank (not shown) and partially recycled after adjustment of the chlorine concentra-tion. The salad falls into a tank (9) filled with drinkable water (chlorine concentrationlower than 0.5 ppm). The rinsing section (13) is also fitted with bumps (12) designedto turn over the leaves and to expose both sides to a UVc tube (24), which preventsany microbial cross-contamination in the rinsing section. The leaves are separatedfrom water onto a second sieve (15). The water is recycled (14) and (17) either tothe first section or to the upstream prewashing. The product is collected into crates(19) or sent directly to the drying system (spin dryer or tunnel).

DRAINING

Excessive free water in packs results in rapid bacterial spoilage mainly at the leaf-film interface (Herner and Krahn, 1973). Draining should result in about 1% residualmoisture compared to the unprocessed salad. Two methods are presently used for this

FIGURE 3.13 Listeria monocytogenes growth on inoculated broad-leaved leaves after drastic(empty bars) or light (closed bars) chlorine disinfecting (Carlin et al., 1996).

4

4.5

5

5.5

6

6.5

7

0 4 7Days at 10 C

log

(cf

u.g

-1)

Not disinfected Disinfected

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36 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

operation: a spin dryer and an air tunnel. Drastic centrifugation results in bruising,so the process was improved using special centrifuges to achieve optimal draining.The centrifugation cycle begins with a soft loading of the fragile leaves followed bya smooth acceleration and a careful discharge of the drained products (Figure 3.15).

Air tunnel drying is a new technique developed in Italy that is currently used inseveral processing plants in Europe and in the United States (Figure 3.16).

The drying tunnel is composed of “cascade” vibrating tables to transport theproduct and a battery of air drying units. The product progression is countercurrentwith both air temperature and dryness. The dryer is microprocessor piloted to optimizeits efficiency. In order to limit cross-contamination by airborne microorganisms, theairflow is filtered and disinfected with a UV tube (250–280 nm).

WEIGHING AND PACKING

The packing room must be clean and refrigerated at 1–2°C and must be separated fromthe washing section. Packing is performed around a vertical tube at the top of whichis the associative weighing machine, an example of which is shown in Figure 3.17.

Salad bits (or any other products) are poured into the infeed funnel (or a vibratingcone) designed to distribute the vegetable chunks evenly into feed buckets, whichrelease them into weighing buckets. The successive bucket system permits a con-tinuous operation of the machine provided the level sensor is not activated. Theweight of plant tissues in all the buckets is transmitted to a computer that calculatesthe best combination to optimize the required weight. Both mean weight and accept-able standard deviation are entered into the computer.

FIGURE 3.14 New washer based on a succession of laminar and torrential flows, includinga regulation of chlorine concentration and a rinsing section (Béguin and Varoquaux, 1996).

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Overview of the European Fresh-cut Produce Industry 37

Vegetable chunks are collected in the collating funnel that feeds the packagingmachine. The first part of the packaging machine is the conforming tube that drivesthe packaging film around the cylindrical tube at an optimal angle. The longitudinalsealing is performed, and the sleeve is driven by two conveying belts. The progressionof the sleeve is guided by a photo cell that reads printed marks on the film. At theend of the tube, the lower part of the sleeve is sealed. Vegetable chunks are then

FIGURE 3.15 Semiautomatic spin dryer optimizing drying conditions for fresh-cut salads.(Courtesy of Rousselet Centrifugation.)

FIGURE 3.16 Air dryer. (Courtesy of Turatti.)

Loading:Low-speed rotationadjustable in conjunctionwith feeding arrangements.

Spinning:Increase speed to effectdrying after feed has beeninterrupted (low-speed spinfor salad products).

Unloading:Low-speed rotation inconjunction with pusherplate function to effectgentle discharge of driedproduct.

Slowrotatingproductdischargeplatform

Double shell

Outlet pipe

Watertight (IP 55)electrical motor

Pusher plate

Articulated lid

Basket

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38 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

released from the weighing buckets. The filled bag may be flushed with nitrogen,carbon dioxide or a binary gas mixture (N2 and CO2) to actively modify the atmo-sphere. The gas injection nozzle is withdrawn, and the upper side of the bag is sealed.

Most minimally processed vegetables in France are packed in bags of polypro-pylene that are 25–40 µm thick. In England and Ireland, a wider range of vegetablesincluding spinach, broccoli, and cauliflower florets is processed. These highly respir-ing commodities must be packed with microperforated films more permeable togases than oriented polypropylene. The permeability of oriented polypropylene isabout 2⋅10−16 and 7.2⋅10−16 mole⋅m−1⋅sec−1⋅Pa for O2 and CO2, respectively. In Europe,professionals use permeance to quantify gas transmission rate instead of permeability.Permeance is the permeability multiplied by the thickness of the film and is expressedin ml of gas per square meter, per day, and per atmosphere or Bar (ml⋅m−2⋅day−1⋅atm−1).

Oriented polyethylene (OPP) was preferred to polyethylene mostly for its bright-ness, crispness, and suitability for machine packing. The permeance of this film issuitable for packaging fresh-cut broad-leaved endive and lettuce provided that thedistribution temperature does not exceed 10°C. This film generates an equilibratedmodified atmosphere (steady state) within the bag that prevents necrosis of saladleaves. Pectinolytic bacteria Pseudomonas fluorescens and Pseudomonas viridiflavaare responsible for soft rot on stored vegetables (Lund, 1983). They are also involvedin the decay of shredded endive mixes (Nguyen-The and Prunier, 1989), but thesebacteria are present in both spoiled and sound packs.

As shown in Table 3.1, broad-leaved leaves inoculated with a heavily concen-trated Pseudomonas marginalis suspension do not develop necrosis when stored inCO2-enriched atmospheres. It is noteworthy that the leaves injected with a filteredculture medium of this bacterium develop the same symptoms. It has been postulatedthat the beneficial effect of CO2 on soft rot induced by P. marginalis is due to the

FIGURE 3.17 Associative weigher. (Courtesy of Yamato.)

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Overview of the European Fresh-cut Produce Industry 39

acidification of cell medium by CO2 dissolution (Siriphanich and Kader, 1986),which in turn, inhibits the enzymatic activity of a pectate lyase produced by thisbacterium (Nguyen-The and Carlin, 1988).

Salads that are highly sensitive to oxidation (such as those including butterheadand iceberg lettuces) are flushed with nitrogen so that residual oxygen within thepacks ranges from 1–3%. Because the atmospheric composition at steady state doesnot depend on the initial gas mixture within the pack (Fishman et al., 1995), theactive modified atmosphere will only accelerate the establishment of a protective atmo-sphere. Some processors introduce CO2 into the pack to obtain 5–10% of CO2 aftersealing.

Shredded carrots and cabbages deteriorate rapidly when stored with excessivelyhigh (over 30 kPa) CO2 and low O2 partial pressures (Carlin et al., 1990a). Barry-Ryan et al. (2000) demonstrated that the spoilage of these products was triggeredby the depletion of O2 more than by the rise in CO2. These detrimental atmosphericconditions result in a physiological disorder and exudation by the carrot tissue. Thissap provides microorganisms, including lactic acid bacteria, with a good growthsubstrate (Carlin et al., 1990a). The spoilage is markedly reduced using a morepermeable film than regular OPP. Microperforated films with a permeance of15,000–20,000 ml O2 (or CO2, because these films are not selective to gas diffusion)markedly reduced spoilage of shredded carrot compared to 35 µm OPP (Carlin et al.,1990b). A too permeable packing film results in a high respiration rate and a rapiddecrease in sugar content of the carrot tissue.

Special products requiring an O2-deprived atmosphere, such as sliced apples orprepeeled fresh potatoes, may be packed in high barrier films using a nitrogen (orother protective gases or gas mixtures) compensated vacuum.

The sell-by date must be stamped on the bag immediately after packaging. Thepacks are placed in cartons and stored in 1–4°C cold room according to the first-in,first-out principle. The expedition platform must also be fitted with a cold lockchamber. The chill chain must be respected and controlled up to the store’s refrig-erated cabinet.

TABLE 3.1Effect of Controlled Atmosphere on the Development of Soft Rot on Inoculated Broad-Leaved Leaves. (From Nguyen-The and Carlin, 1988.)

Controlled Atmosphere

% CO2 % O2

Inoculum

H2O (Control) Ps. m

Filtrate Medium Ps. m

40 10 0 0 0 20 10 0 0 (+) 0 22 0 ++ ++

Ps. m: Pseudomonas marginalis; 0: no spoilage; (+): no browning, slight soft rot;++: browning soft rot.

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40 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

CONCLUSION

NEW PRODUCTS

About 10 years ago, it was thought that diversification would boost the sales offresh-cut commodities. The development of fresh-cut fruit salad was one of the firstideas.

Fresh-cut Fruits

In the first attempt to manufacture these products, the only additives or preservativesused were ascorbic or erythorbic acids. The shelf life of these fruit salads in juicewas short (five days), and on the sell-by date, they were highly contaminated byyeast (107–108 cfu⋅g−1). Production was stopped after one year or so. More recently,Belgian and Dutch processors have manufactured fresh fruit salads with syrupstabilized with sorbate (to prevent yeast growth), ascorbic acid (as an antibrowningagent), and calcium chloride (to reduce texture breakdown). Of these chemicals,only sorbate is controversial. The stabilization of fresh fruit salads packed withoutany liquid (syrup or fruit juice) requires only a few ppm of sorbate, because onlythe first layers of damaged cells must be protected (Varoquaux and Varoquaux, 1990).Dry fruit chunks must be packed in almost airtight containers and under anoxia inorder to prevent browning. Nitrous oxide (N2O) and carbon dioxide (CO2) are moreefficient than N2. When the fruit chunks are immersed, the sorbate concentrationmust be much higher, from 350 ppm (sorbic acid) when distributed between 0 and4°C to 1000 ppm when stored at ambient temperature (20°C). The organolepticqualities (flavor, firmness, and appearance) are better protected in the “dry” versionthan in the immersed one.

Vegetable Mixes

Stir fry and other preparations to be served with meat are increasingly popular inEurope. These products, already developed in the United States, are being adaptedto the European market.

Niche Products

Mushrooms, including truffles, and local salad mixes, such as “mesclun” (a Medi-terranean salad mix), are examples of available niche products.

NOVEL PROCESSING TECHNIQUES

Automatic Trimming

The major fresh-cut French companies have developed automatic trimming machines.In one such machine, the stump of a broad-leaved plant is seized by a claw thatspins, thus flattening the salads by centrifugal force. The green extremity of theouter leaves is torn off by static knives placed at an optimal distance and angle fromthe rotation axis. This machine has been considerably improved and is in operation

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Overview of the European Fresh-cut Produce Industry 41

at three processing plants in France and England. In another machine (no longer inuse), the knives rotated around the salads. A study has also been conducted in whichthe salad is trimmed with a water jet guided by computerized image analysis. Theproject did not result in an operable automatic trimmer.

Chlorine-Free Fresh-cut Commodities

As previously mentioned, the use of chlorine for disinfecting in the food industryis controversial and forbidden for organic food. The efficiency of ozone, organicacids, peracetic acid and hydrogen peroxide, the glucose oxidase lactoperoxidasesystem, air ionization, and other techniques is under investigation. Gamma irradia-tion and accelerated electrons are effective on shredded root vegetables but resultin a dramatic texture breakdown when applied to green salads.

Modified Atmosphere Packaging (MAP)

Optimization of actively modified atmosphere and film permeance is being studiedto make MAP more effective. This includes the use of high oxygen concentrationsand noble gases.

Prevention of Temperature Abuse

Time-temperature indicators (TTI) have been used on cartons in France to detecttemperature abuse during transport and distribution. The detrimental effect of tem-perature abuse may also be buffered by the packaging of fresh-cut bags or punnetsin polystyrene boxes instead of cartons. This technique has been used in France, butits financial and environmental costs were high because the isothermal containercould not be recycled.

REFERENCES

Anonymous. 1986. “L’industrie des légumes frais prêts à l’emploi, dits légumes de la 4èmegamme.” Strasbourg, France: Arist Alsace, pp. 1–131.

Anonymous. 1988. “Guide des bonnes pratiques hygiéniques concernant les produits végétauxprêts à l’emploi, dits “de 4ème gamme,” approuvé le 1er Août 1988 par le directeurgénéral de la concurrence, de la consommation et de la répression des fraudes. Bulletinofficiel de la concurrence, de la consmmation et de la répression des fraudes, 17:221–233.

Anonymous. 1993. “Arrêté du 22 Mars 1993 relatif aux règles d’hygiène applicables auxvégétaux et préparations de végétaux crus prêts à l’emploi à la consommationhumaine.” Journal Officiel de la République Française. 75: 5586–5588.

Anonymous. 1996. “Guide de bonnes pratiques hygiénique-végétaux crus prêts à l’emploi.”Les éditions du Jounal Officiel, Paris, 71.

Barry-Ryan C., Pascussi J.-M. and O’Beirne D. 2000. “Quality of shredded carrots as affectedby packaging film and storage temperature.” J. Food Sci., 65: 726–730.

Béguin G. and Varoquaux P. 1996. “Procédé de lavage et de désinfection de feuilles de légumestels que des salades.” French Patent number 96 08644.

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42 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Béguin G., Clays G. and Delavet C. 1995. “Procédé et dispositif de découpe de légumes etde fruits.” French Patent number 95 04674.

Bolin H.R. and Huxsoll C.C. 1991. “Effect of preparation procedures and storage parameterson quality retention of salad-cut lettuce.” J. Food Sci., 56: 60–62, 67.

Bolin H.R., Stafford A.C., King A.D. Jr. and Huxsoll C.C. 1977. “Factors affecting the storagestability of shredded lettuce.” J. Food Sci., 42: 1319–1321.

Carlin, F., Nguyen-The C., Cudennec, P. and Reich M. 1989. “Microbiological spoilage offresh, ready-to-use grated carrots.” Science des Aliments. 9: 371–386.

Carlin F., Nguyen-The C., Chambroy Y. and Reich M. 1990a. “Effects of controlled atmo-spheres on microbial spoilage, electrolyte leakage and sugar content on fresh, ‘ready-to-use’ grated carrots.” Int. J. Food Sci. Technol., 25: 110–119.

Carlin F., Nguyen-The C., Hilbert G. and Chambroy Y. 1990b. “Modified atmosphere pack-aging of fresh ‘ready-to-use’ grated carrots in polymeric films.” J. Food Sci., 4:1033–1038.

Carlin F., Nguyen-The C. and Morris C.E. 1996. “The influence of the background microfloraon the fate of Listeria monocytogenes on minimally processed fresh broad leavedendive (Cichorium endivia var. latifolia).” J. Food Protection, 59: 698–703.

Fishman X., Rodov V., Pertz J. and Ben-Yehoshua S. 1995. “Model for gas exchange dynamicsin modified atmosphere packages of fruits and vegetables.” J. Food Sci., 60:1078–1087.

Forney C.F. and Austin R.K. 1988. “Time of day at harvest influences carbohydrate concen-tration in crisphead lettuce and its sensitivity to high CO2 levels after harvest.” J. Am.Soc. Hort. Sci., 113: 338–340.

Goupy P., Varoquaux P., Nicolas J. and Macheix J-J. 1990. “Identification and localizationof hydroxycinnamoyl and flavonol derivatives from endives (Cichorium endivia L.cv Géante maraichère) leaves.” J. Agric. Food Chem., 38: 2116–2120.

Goupy P., Macheix J-J., Nicolas J. and Varoquaux P. 1994. “Partial purification and charac-terization of endive (Cichorium endivia L.) polyphenoloxidase.” Sci. Alim., 14:751–762.

Herner R.H. and Krahn T.R. 1973. “Chopped lettuce should be kept dry and cold.” YearbookProd. Mark. Assoc., p. 130 in Bolin et al. (1977).

International Commission on Microbiological Specifications for Foods (ICMSF). 1986.Microorganisms in Foods 2: Sampling for Microbiological Analysis: Principles andSpecific Applications. 2nd Edition. Blackwell Scientific Publications, Great Britain.

International Commission on Microbiological Specifications for Foods (ICMSF). 1988.Microorganisms in Foods 4: Applications of the Hazard Analysis Critical ControlPoint (HACCP) System to Ensure Microbiological Safety and Quality. 2nd Edition.Blackwell Scientific Publications, Great Britain.

Kuczinski A., Varoquaux P. and Souty M. 1993. “Reflectance spectra of ‘ready-to-use’ appleproducts for determination of enzymatic browning,” Int. Agrophysics, 7: 85–92.

Lund B.M. 1983. “Bacteria spoilage.” In Post-Harvest Pathology of Fruits and Vegetables,C. Dennis (ed.), London, Academic Press, pp. 218–257.

Nguyen-The C. and Carlin F. 1988. “Altérations microbiologiques des légumes prêts àl’emploi.” Deuxième Conférence Internationale sur les Maladies des Plantes. Bor-deaux, 8–10 Nov. Proceedings 1: 743–750.

Nguyen-The C. and Carlin F. 2000. “Fresh and processed vegetables.” In The MicrobiologicalSafety and Quality of Food, B. M. Lund, T. C. Baird-Parker and G. W. Gould (eds.),Gaithesburg, Aspen Publisher, pp. 620–684.

Nguyen-The C. and Prunier J.P. 1989. “Involvement of Pseudomonas in ‘ready-to-use’ saladsdeterioration.” Int. J. Food Sci. Technol., 24: 47–58.

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Sabino J. 1990. “Contribution de l’acheteur aux innovations: Le cas de la quatrième gammefruits et légumes frais,” DESS, Univ. Grenoble II, France, pp. 1–64.

Scandella D. and Leteinturier J. 1989. “La quatrième gamme.” Paris, France: CTIFL ed.,pp. 1–69.

Siriphanich J. and Kader A.A. 1986. “Changes in cytoplasmic and vacuolar pH in harvestedlettuce tissue as influenced by CO2.” J. Am. Soc. Horticult. Sci., 111: 73–77.

Varoquaux P. and Varoquaux F. 1990. “Les fruits de quatrième gamme.” Arb. Fruit., 3: 35–38.Varoquaux P. and Wiley R.C. 1996. “Biological and biochemical changes in minimally

processed refrigerated fruits and vegetables.” In Minimally Processed RefrigeratedFruits and Vegetables, R.C. Wiley (ed.), New York: Chapman & Hall, pp. 226–268.

Varoquaux P., Lecendre I., Varoquaux F. and Souty M. 1990. “Change in firmness of kiwifruitafter slicing.” Sci. Alim., 10: 122–139.

Varoquaux P., Mazollier J. and Albagnac G. 1996. “The influence of raw material character-istics on the storage life of fresh-cut butterhead lettuce.” Postharvest Biol. and Tech-nol., 9: 127–139.

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Safety Aspects of Fresh-cut Fruits and Vegetables

William C. Hurst

CONTENTS

Food Safety Question Fresh-cut Risk FactorsControl PointsSamplingThe HACCP ApproachWhy Use HACCP?What HACCP Is NOTOrigin of HACCPPrerequisites of HACCPGood Agricultural Practices (GAPs)Standard Operating Procedures (SOPs) Good Manufacturing Practices (GMPs)Sanitation Standard Operating Procedures (SSOPs)Comprehensive Plant Sanitation

General PrinciplesFacility (Environmental) Sanitation Equipment Sanitation Sanitation Audit

Visual InspectionMicrobiological MonitoringTools to Use

Pest ControlEmployee Hygiene Practices

Developing an HACCP ProgramAssemble the HACCP TeamHACCP TrainingDefine the ProductDevelop a Flow DiagramPerform a Hazard Analysis (Principle 1)

4

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Identify Critical Control Points (Principle 2)Set Critical Limits (Principle 3)Establish Monitoring Procedures (Principle 4)

What Will Be Monitored?When Will Monitoring Be Performed?How Will Monitoring Be Performed?Who Will Perform the Monitoring?Statistical Monitoring for Process ControlIntegrating SPC and HACCPSPC Monitoring of CCPs

Establish Corrective Actions (Principle 5)Establish Verification Procedures (Principle 6)

SPC Validation/Verification of a ProcessEstablish Record-Keeping Procedures (Principle 7)SummaryReferences

FOOD SAFETY QUESTION

As food safety continues to be a worldwide public health issue, epidemiologicalstudies have shown a significant increase in the number of produce-related foodborneillnesses over the past three decades. The Centers for Disease Control and Prevention(CDC) have reported that the mean number of outbreaks associated with fruits andvegetables more than doubled from 1973 to 1987 (4.3 per year) and again from 1988to 1991 (9.75 per year).

Salmonella

spp. were the most common etiological agentslinked to these outbreaks. During 1995 alone, major outbreak investigation linkedinfections with

Salmonella

serotype Stanley to alfalfa sprouts,

Salmonella

Hartfordto unpasteurized orange juice,

Shigella

spp. to lettuce and green onions,

Escherichiacoli

O157:H7 to lettuce, and hepatitis A virus to tomatoes (Tauxe, 1997). Morerecently in the United States, The Center for Science in the Public Interest rankedfresh produce the fourth highest cause of all food illness since 1990 (Figure 4.1),behind seafood, eggs, and beef. This data excluded bagged salads, fruit salad, orother processed produce. Sprouts and lettuce were the most frequent culprits. In arelated study, labeled “multi-ingredient foods,” 14 outbreaks over the 10-year period(1990–2000) were attributed to bagged salads as well as salad bars and processeditems not devoted exclusively to produce items (Scruton, 2000). The importance offresh produce as a vehicle for pathogen transmission and those specific pathogensepidemiologically implicated in fresh produce-related diseases have been extensivelyreviewed and documented (Doyle, 1990; Nguyen-The and Carlin, 1994; Beuchat, 1996;Beuchat and Ryu, 1997; Tauxe et al. 1997; DeRoever, 1999; Gillian et al., 1999).

Media attention to fresh and fresh-cut produce has heightened consumer aware-ness of produce safety. In 1998, a Fresh Trends survey, conducted by

The Packer

magazine, found that 60% of consumers are more concerned today than they werea year ago about

Salmonella

and other bacteria in produce. In response to consumerconcerns, major retailers (e.g., Albertson’s and Safeway) and foodservice restaurants

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(e.g., Taco Bell and Burger King) have begun programs requiring their suppliers(growers) to have independent third-party inspections of their farms to certify thatfruits and vegetables are being grown, harvested, and handled using good agriculturalmanagement practices (Hodge, 1998; Beers, 1999). Processors of fresh-cut producehave long understood their responsibility for providing a microbiologically safe,high-quality product to the consumer. They have always taken a proactive attitudetoward safety. Within two years of establishing an industry association of processorsand suppliers in 1987, a sanitation task force was commissioned to develop modelsanitary guidelines for the membership. The culmination of this work was a publicationentitled “Recommended Sanitary Guidelines for the Produce Processing Industry”that was released in 1992. This document set forth basic sanitary Good Manufac-turing Practices (GMPs) and standardized processing procedures to ensure consistentquality and improved a processor’s credibility with local, state, and federal foodinspectors for ensuring a safe product to the consumer (Hurst, 1992).

Despite the many technological and educational advances within the fresh-cutindustry in its short history, the challenge remains how to best ensure product safety.The purpose of this chapter is to review some control measures for safety and providea preventative strategy that has proven most advantageous for minimizing food safetyhazards.

FRESH-CUT RISK FACTORS

Food hazards may be regarded as microbiological, chemical, or physical in nature.However, because microbiological safety is the major issue of concern in the fresh-cut industry, it will be the focus of this chapter. Many factors may be involved inthe epidemiology of produce-associated disease. Risks for fresh-cut can be dividedinto two categories. One category concerns the factors or conditions contaminatingfresh produce with indigenous pathogens during cultivation or at harvest. These have

FIGURE 4.1

Cases of foodborne illnesses related to seafood (circles), eggs (solid brokenlines), beef (solid line), and produce (squares) for 1990–1997. (Data reprinted with permissionfrom the Center for Science in the Public Interest. Data for 1998–2000 are not yet complete.)

1990 1991 1992 1993 1994 1995 1996 1997

40

35

30

25

20

15

10

5

0

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been addressed by Hedberg et al. (1994) and Tauxe et al. (1997). They include pooragronomic practices, use of contaminated water for crop irrigation or mixing chem-ical sprays, application of improperly composted manure as fertilizer, and lack oftraining among field workers on good personal hygiene. Poor sanitary control duringpostharvest handling activities is another mechanism for pathogen contamination tofresh produce. DeRoever (1999) reported that two large cases of salmonellosisoccurred in 1991 and 1993 due to fresh tomato consumption. The suspect tomatoeswere epidemiologically linked to a single tomato packinghouse. Although the exactcontaminating point was never pinpointed, possible sources included improperlycleaned bins, buckets, and trucks used for transporting tomatoes from the field tothe packinghouse, cross-contamination of tomato dump tank water, poor personalhygiene among the employees, and/or improperly cleaned equipment that routinelywent a full season without being disassembled for cleaning.

A second category of microbiological risk is the cutting or slicing operation inthe fresh-cut plant. Internal tissue of fresh produce is normally protected from micro-biological invasion by waxy outer skins and peels. However, cutting circumventsthis physical barrier, allowing juices to leak from inner tissues onto the surfaces offruits and vegetables. These juices contain nutrients for accelerating microbiologicalgrowth. Together with an increase in exposed surface area, large microbiologicalpopulations, including potentially higher human pathogen levels, develop on cutproduce items (Brackett, 1987; Garg et al., 1990). Key microbiological risks of fresh-cut produce have been determined (Hurst, 1995; Fain, 1996; Zagory and Hurst,1996) and include the following: there is no kill step (such as cooking) in the processto eliminate potential human pathogens; several pathogens (e.g.,

Listeria monocy-togenes

and

Aeromonas hydrophila

) are psychotrophic and can grow at temperaturesused to store fresh-cut products; the longer shelf life (10–14 days) that is nowcommon, due to good temperature control and sophisticated packaging, may providesufficient time for pathogen growth; modified atmospheres suppress the growth ofspoilage organisms, but certain pathogens (

Listeria monocytogenes

) survive and mayactually thrive under these conditions; and unlike traditionally processed (cannedand frozen) fruits and vegetables, fresh-cut produce is consumed raw.

CONTROL POINTS

Contamination of human pathogens on fresh produce may occur at any stage duringits production, harvesting, handling, processing, storage, or distribution to the con-sumer. Growers, packers, and shippers of fresh produce have recently been provideda guide on how to minimize microbiological safety hazards during agriculturaloperations. This document (CFSAN, 1998) sets forth Good Agricultural Practices(GAPs) for producers to implement in their farm facilities. In addition, it focuseson three educational messages: to increase awareness of the common microbiolog-ical hazards in fruit and vegetable production, to stress prevention of contaminationover corrective actions once contamination has occurred, and to establish a formatfor developing a system of accountability of sanitary practices at all levels of the

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agricultural and packinghouse environment. The GAPs document identifies whereand how to control microbiological risks between the field and the fresh-cut pro-cessing plant.

While GAPs address field sanitation practices, Good Manufacturing Practices(GMPs) provide the framework for minimizing product contamination in the pro-cessing plant. FDA mandates GMPs for all food handling establishments. Fresh-cutprocessors must have a comprehensive sanitation program, encompassing the facility,equipment, and personnel, as part of their quality assurance system. GMPs are thefirst line of defense in controlling pathogen buildup in the product or environmentof a fresh-cut processing plant.

SAMPLING

To verify GMP control, food processors have traditionally used end-product testingto evaluate microbiological quality and safety of their foods. Microbiological testingis performed on a given number of samples collected from a production run, or lot,of product. When employing a sampling scheme to test for the presence of micro-organisms, samples must be drawn in such a manner that the microbiological qualitydetermined by the results is an accurate representation of the microbiological char-acter of the lot. However, as demonstrated in Figure 4.2, microorganisms are notrandomly distributed throughout the food. Instead, cells aggregate to form a conta-gious distribution (Jarvis, 1989), a type of nonhomogenous distribution. Several types

FIGURE 4.2

Possible types of spatial distribution of microorganisms in food.

σ

2

, variance;

µ

, mean. (Reprinted with permission from Jarvis, 1989.

Statistical Aspects of the Microbio-logical Analysis of Foods

, Volume 21. Elsevier Science Publishers.)

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of probability distributions can be utilized to deal with nonrandom data. The probabilitydistribution used to describe the statistically based attribute sampling plans for themicrobiological examination of foods is the binomial (ICMSF, 1986). The problemof using attribute sampling (based on the binomial distribution) in looking for patho-gens in fresh-cut products is that when the defective level

p

(e.g., pathogen) is verylow, a very large number of fresh-cut samples

n

would have to be tested to give a highprobability that one defective unit would be found. Figure 4.3 illustrates the futilityof this procedure. It is a graph of the probability of detecting at least one defectivesample (pathogen positive) for various sample sizes when the level of the defectiveunits is 0.1% and 0.01%. The lower curve, which represents the probability of detectionwhen there is a 0.01% level of defective units, does not even approach 0.5 probabilityat a sample size of 5000 units! On the other hand, a probability approaching one isshown when the sample size approaches 5000 at a 0.1% level of defective units. Asthe number of samples tested increases, so does our confidence in the results, but, sotoo does the cost of sampling. Thus, if close to 100% inspection must be employed todetect low levels of pathogens in a fresh-cut product, clearly, sampling is an inadequatemeans of assuring safety in products leaving the processing plant. Because sampling

FIGURE 4.3

Probability of detecting a defective sample (positive for pathogens) based onthe % defectives in a lot. (Reprinted with permission from Toledo, 2000.)

1.0

0.8

0.6

0.4

0.2

0

0.1% defective

0 1000 2000 3000 4000 5000 6000

0.01% defective

Number of Samples

Pro

bab

ility

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techniques never provide a good enough answer for safe microbiological control,we must look to other approaches.

THE HACCP APPROACH

As indicated above, monitoring a finished food product is no guarantee of safety becauseunsafe samples may escape detection. What is needed is a more focused approachtoward controlling food safety. Such a program is the Hazard Analysis Critical ControlPoint (HACCP) concept. HACCP is a structured approach to the identification, assess-ment of risk (likelihood of occurrence and severity), and control of hazards associatedwith a food production process or practice. HACCP addresses the root causes of foodsafety problems in production, storage, transportation, etc., and is preventative (FDA,1994). It aims to identify possible problems before they occur and establish controlmeasures at stages in production that are critical to product safety. One of the purposesof HACCP is to design safety into the process, thereby reducing the need for extensivemicrobiological testing of in-line samples and finished product (Silliker, 1995).

Design and implementation of a HACCP system involves following seven basicprinciples or steps (Stevenson and Bernard, 1999).

Step 1

: Conduct a hazard analysis. Flow diagram the steps of a processto determine where significant hazards exist and what control measuresshould be instituted.

Step 2

: Determine Critical Control Points (CCPs) required to control theidentified hazards. CCPs are any steps where hazards can be prevented,eliminated, or reduced to acceptable levels.

Step 3

: Establish Critical Limits (CLs). These are specifications (target valuesand tolerances) that must be met to insure that CCPs are under control.

Step 4

: Establish procedures to monitor CCPs. These are used to adjustthe process to maintain CCP control.

Step 5

: Establish corrective actions to be taken when monitoring indicatesa deviation from an established critical limit.

Step 6

: Establish verification procedures for determining if the HACCPsystem is working correctly.

Step 7

: Establish effective record-keeping procedures that document theHACCP system.

HACCP for the fresh-cut industry must be built around a series of preservativefactors (hurdles) to control pathogen growth, because there is no definitive kill stepin the processing operation. Hurdle technology uses a combination of suboptimalgrowth conditions in which each factor alone is insufficient to prevent the growthof pathogens, but when combined in additive fashion give effective control (Gorrisand Tauscher, 1999). Fresh-cut hurdles include purchasing produce from certifiedgrower/packers, implementing comprehensive plant sanitation programs, using numer-ous antimicrobial agents (Beuchat, 1998) in the wash water, using modified atmo-sphere packaging techniques (Gorris and Tauscher, 1999), and following consistentlow temperature management.

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WHY USE HACCP?

Perhaps the foremost reason for implementing HACCP into a fresh-cut operation isto be ready for impending government regulations. HACCP has become a mandatoryprogram for several food industries in recent years. FDA placed the seafood industryunder HACCP at the end of 1997 (FDA, 1997), and the USDA required all meatand poultry establishments to be under HACCP by January 25, 2000 (FSIS, 1996).Most recently, FDA issued a possible fresh juice HACCP mandate, which is still underreview (FDA, 1998). FDA has revised its 1999 Food Code, announcing that all retailbusinesses that handle, distribute, or process food shall implement a food safety planbased on the HACCP concept (FDA, 1999). The message is clear—all food processorswill have HACCP in their future (Stier and Blumenthal, 1995). HACCP undoubtedlywill be the food safety system of the future for regulatory use (Corlett, 1998).

HACCP is a proven, cost-effective method of maximizing food safety, because itfocuses on hazard control at its source. It offers systematic control by covering allaspects of production and handling from raw materials to consumer preparation.HACCP builds customer confidence that food safety is being effectively managed at afresh-cut operation. Because of its stringent controls, HACCP will bring about improve-ments in product quality. It demonstrates where to target resources to reduce risks.HACCP implementation will reduce losses from recalled or reworked product. HACCPcomplements total quality management because it offers continuous problem prevention.

WHAT HACCP IS NOT

Although HACCP implementation may lead to product quality improvement, itshould be a distinctly separate program, not incorporated with quality control in afresh-cut operation. HACCP is a food safety system and should focus solely onsafety issues (Scott, 1993). For example, HACCP would be designed to prevent

E.coli

O157:H7 contamination in fresh-cut lettuce but would not guarantee the absenceof brown leaves in a 2-lb bag (Hurst, 1995). In the early days of HACCP development,too many areas were designated as “critical,” causing the overlap of safety and qualitypoints. The result was frustration and overwork among personnel who were respon-sible for monitoring and documenting these areas. Mixing safety and quality aspectsin the same plan caused a dilution of the really critical areas and failures of manyHACCP plans.

HACCP is not a substitute for the FDA’s Sanitary GMPs (Good ManufacturingPractices). In fact, effective sanitation must be a prerequisite for successful imple-mentation of a plant HACCP program. Sanitary procedures may, however, becomeincorporated as a control tool in HACCP plans to prevent a hazard from becominga reality. For example, scarred cutting boards on a lettuce trim line may have shownto be a niche for microbiological pathogens based on equipment audits, if carefulsanitation does not exert control over this area.

Also, HACCP plans, unlike GMPs, are not designed to cover all areas of sanitarycontrol in a food operation. Instead, they narrowly focus on specific areas wherehazards might be introduced. All GMP requirements are equal from a regulatoryperspective. So, dust is filth under GMPs, and its presence on equipment is a violation.

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However, under HACCP, control of dust is a sanitary step but is not

critical

, becauseits presence is unlikely a safety hazard.

ORIGIN OF HACCP

The HACCP concept was originally conceived by the Pillsbury Company in collab-oration with the U.S. Army Natick Research and Development Laboratories andNational Aeronautics and Space Administration to develop safe food for astronautsin the early 1960s. Pillsbury found that the only way to give 100% assurance thatspace foods were free of microbiological pathogens was to test all products. Finishedproduct testing was impractical, because most of the product was used up duringthe destructive nature of the test procedures. A better method of food safety assurancewas obviously needed, and this is how HACCP was born (Stevenson, 1993).

PREREQUISITES OF HACCP

As HACCP is integrated into food industry management systems, it becomes evidentthat HACCP cannot exist as a stand-alone program. Sperber et al. (1998) point outthat HACCP cannot be successfully applied in a vacuum, but rather, it must besupported by a strong foundation of prerequisite programs. While not a formal partof HACCP, prerequisite programs must be developed and implemented in a foodprocessing operation before attempting to put a HACCP plan in place.

Prerequisite programs are written, implemented procedures that address opera-tional conditions and provide the documentation to help an operation run more smoothlyto maintain a comprehensive food-safety assurance program. Fresh-cut processorsshould develop written prerequisite programs for the following operations: raw mate-rial receipt and storage; wash water quality; equipment maintenance; productioncontrols for grading, washing, cutting, drying, and packaging; temperature and micro-biological controls; chemical control; sanitary control for the facility, equipment, andemployees; product coding and traceability; recall procedure control; and finishedproduct storage and distribution control.

GOOD AGRICULTURAL PRACTICES (GAPs)

In 1997, President Clinton announced his Food Safety Initiative amid public andmedia pressure to improve safety in the U.S. food supply. An immediate result wasthat the FDA in conjunction with the USDA published a user’s manual for the freshproduce industry entitled “A Guide to Minimize Microbiological Food Safety Haz-ards for Fresh Fruits and Vegetables.” This document, which is not regulatory,identifies potential sources of microbiological contamination for fruits and vegeta-bles during production and handling at the farm level and provides suggestions on“good agricultural practices” to minimize these hazards (CFSAN, 1998). Specifi-cally, it addresses potential contamination from water sources, fertilizer use (manureor compost), worker health and hygiene, and field and packingshed sanitation, andcalls for the development of trace-back procedures for fresh produce.

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STANDARD OPERATING PROCEDURES (SOPs)

The heart of any prerequisite program are the Standard Operating Procedures (SOPs).SOPs are written references used to describe a specific sequence of events necessaryto perform a task (Harris and Blackwell, 1999). In other words, they are step-by-step instructions that outline how an operation is to be carried out. SOPs must bewritten for both safety and nonsafety operational tasks. For example, a detailedprocedure should be written for monitoring and maintaining correct disinfectantlevels of fresh-cut wash water to insure product safety. Likewise, a specific SOP forchecking product temperature should be developed that gives instruction on whereand how often to perform the task to maintain product quality and shelf life. SOPsare used to assure that critical processing steps are accomplished and can also beused to train employees.

GOOD MANUFACTURING PRACTICES (GMPs)

GMPs are the minimum sanitary and processing requirements necessary to insure theproduction of wholesome food (Harris and Blackwell, 1999). FDA requirements forGMPs are listed in Title 21, Part 110 of the Code of Federal Regulations. GMPs arewritten for the following plant areas: building and facilities, equipment and utensils,employee practices, pest control, production and process controls, and warehousingpractices. GMPs are broadly written, general in nature, and not intended to be plantspecific. GMPs can be used to explain tasks that are part of many jobs (e.g., GMPsare written for personal hygiene and dress regardless of job title, management, pro-duction, quality assurance, maintenance, etc.).

GMPs differ from HACCP in a number of ways. First, they are not designed tocontrol specific hazards; second, they do not provide methods for monitoring haz-ards; and third, they do not require specific record-keeping procedures. GMPs are notused to establish deviation limits and do not describe corrective action requirements.

SANITATION STANDARD OPERATINGPROCEDURES (SSOPs)

Sanitation SOPs focus more narrowly on specific procedures that allow a fresh-cutprocessing plant to achieve sanitary process control in its daily operation. SSOPscan be categorized as two types. SSOPs refer to the sanitary procedures used priorto the start of production (preoperational sanitation). OSOPs (Operational SanitationOperating Procedures) refer to sanitary actions taken during production to preventproduct contamination or adulteration (Stevenson and Bernard, 1999). Preopera-tional sanitary procedures are written references that describe the cleaning of equip-ment, utensils, the processing line, and the facility area. Specific instructions mustinclude a description of equipment disassembly, use of approved chemicals accord-ing to label directions, cleaning techniques, reassembly of equipment, and properuse of approved sanitizers.

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Operational SOPs are sanitary practices that must be performed and documenteddaily to validate that fresh-cut product safety was maintained during production.There are five key elements to writing an OSOP. They include a written action planidentifying the task, the frequency of the task, the person responsible for the task,the person responsible for verifying the activity, and corrective actions taken if theexpected outcome is not met. By establishing effective prerequisite programs priorto designing and implementing HACCP, the number of critical control points (CCPs)intended in a plan for fresh-cut plants may be reduced. HACCP becomes more “userfriendly” and manageable because resources can be concentrated on the hazardsassociated with the product, not the processing environment. If SSOPs and/or OSOPsare included as part of the HACCP plan, they must lend themselves to all aspectsof a CCP, including established critical limits and monitoring, corrective action,record keeping, and verification procedures.

COMPREHENSIVE PLANT SANITATION

GMPs serve as the basis for food plant sanitation programs that are mandated bythe FDA. Comprehensive plant sanitation must address three areas: facility environ-ment, processing equipment, and all employees (Zagory and Hurst, 1996). A desig-nated person (sanitarian) who has satisfactorily completed a certified food sanitationprogram should be in charge of writing formal plans and procedures that are to becollected into a sanitation manual. The components of this manual should includethe elements listed below.

G

ENERAL

P

RINCIPLES

1. Specific plant areas (e.g., employee restrooms, break rooms, waste areas,processing and warehouse room floors) must be cleaned and sanitizeddaily based on written procedures. Idle equipment, packaging supplies,and pallets should be removed from the processing area prior to cleanup.Storage and structural facilities, coolers, and other plant areas should becleaned and sanitized on a frequent basis that is best determined by visualand microbiological auditing. Date, time, agent used, person doing thetask, and person verifying the activity must be documented.

2. All processing equipment must be cleaned and sanitized based on written,documented, and verified procedures. The sanitarian would be responsibleby assembling all SSOPs, OSOPs, and SOPs for GMPs into a sanitationmanual.

3. Outside property, such as the exterior building walls, grounds, and land-scaping should be included in the sanitation program.

4. A daily sanitation log of preoperational and operational sanitary condi-tions must be kept on file. Production personnel should be trained to reportany unsanitary conditions to their supervisor immediately.

5. A container identification program must be initiated. Containers for prod-uct and those for waste must be clearly differentiated and never exchanged.

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Employee training should focus on the correct sorting, handling, andstorage of all finished, in-process, and waste fruit and vegetable products.

6. The sanitarian should design and implement continuous education pro-grams on sanitary practices for new and existing employees.

F

ACILITY

(E

NVIRONMENTAL

) S

ANITATION

The environment of a fresh-cut processing facility can be an ideal source of micro-biological contamination. High humidity and numerous structural niches encouragemicrobiological buildup, which is often overlooked during regular equipment clean-ing and sanitizing (Gabis and Faust, 1988). Of greatest concern is the presence ofthe environmental pathogen

Listeria monocytogenes

. Fresh-cut produce facilities canserve as a breeding ground for

L. monocytogenes

because they are cold and wet, andthis microorganism is found on incoming fresh vegetables from the field. To precludeit from gaining a foothold in the cold storage or processing rooms, an environmentalsanitation program must be established (Ryser and Marth, 1991; NACMCF, 1991).Information on where to clean and what to use can be found in the master sanitationschedule of Table 4.1. Dry-cleaning methods should replace wet cleaning in thefinished product cooler, packaging-material and chemical-storage areas, andemployee locker areas of the plant. Cleaning materials, such as dried, compressedair, and tools, such as brushes, brooms, and vacuum cleaners, can be used to removedirt, dust, and other debris likely to attract rodents.

Routine monitoring for the possible presence of

L. monocytogenes

should bedone at its commonly known niches in the plant. Generally, these are nonproductcontact surfaces such as wet floors, drains, cleaning aids, condensation off structuralcomponents, condensation catch pans under refrigeration units, etc. Verification for

L. monocytogenes

presence can be achieved by microbiological testing of air, water,and ice supplies used in the facility. These data are useful in determining sites wherebuildup and movement of airborne and other microorganisms occur throughout thefacility.

E

QUIPMENT

S

ANITATION

Processing equipment should be designed to avoid microbiological growth niches(inaccessible areas that trap product residue and water). Specific directions shouldbe written for disassembling each piece of equipment. Most importantly, sanitationcrews must be trained to follow these procedures during cleaning and sanitizing.Some equipment manufacturers provide written instructions and/or videotapes forcleaning and sanitizing equipment (Shapton and Shapton, 1991). Detailed cleaningprocedures should be documented for each functional piece of equipment as part ofthe total sanitation program. A preoperational sanitation schedule is given in Table 4.2for an Urschel CC carrot shredder.

Operational SOPs generally focus on employee hygiene, product handling,unsanitary conditions that may arise during production, and a mid-shift cleanup.Table 4.3 gives an SOP example for hand washing/sanitizing.

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TABLE 4.1Processing Plant Environmental Sanitation Master Schedule

AreaSanitation Method Tool

CleaningMaterials Frequency

Walls Foam, brush, rinse

Soft nylon brush

Chlorine-quat- based cleaner

Once/month

Ceiling Foam, brush, rinse

Nylon brush, high-pressure machine

Chlorine-quat- based cleaner

Once/month

Floors Wash, rinse Hard bristle broom, hose

Chlorine-quat- based cleaner

Daily

Doors Foam, scrub Scouring pad, cloth

Chlorine-quat-based cleaner

Once/quarter

Plastic curtains Scrub, rinse Scouring pad Chlorine-quat-based cleaner

Once/quarter

Overhead pipes, electrical conduits, structural beams

Foam, brush Brush, bucket, high water pressure machine

Chlorine-quat-based cleaner

Once/month

Hoist Wipe, clean Cleaning pad Water, light soap Once/quarterOverhead light fixtures

Wipe, clean Cleaning pad Water, light soap Once/quarter

Refrigeration coils Rinse, sanitizer High-pressure hose

Water, sanitizer Once/quarter

Chillers Scouring Scouring pad Acid cleaner As needed/auditAir distribution filter Soak Plastic bins Chlorine-based

soapOnce/quarter

Drains, trench Clean, flood Soft nylon brush, 50 gallon container

Chlorine-based soap quaternary ammonium sanitizer

Daily

Grids Brush Nylon brush, high-pressure machine

Chlorine-based soap

Once/week

Waste, dumpster areas

Foam, brush, rinse

Nylon brush, high-pressure foam machine

Heavy-duty chlorine-based cleaner

Daily

Employee break rooms/bathrooms

Wash, rinse Nylon brush, washcloths

Chlorine-based soap

Daily

Maintenance areas Scrub, rinse Nylon brush Degreasing agent Once/month

Source:

Reprinted with permission from Zagory and Hurst, 1996,

Food Safety Guidelines for theFresh-cut Produce Industry,

IFPA.

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TABLE 4.2Sanitation Standard Operating Procedure (SSOP) for an Urschel Machine

Date___________

Fresh-cut, Inc.

Page 1 of 2Standard Operating Procedure

Pre-Op SSOP #8 Revision #________ ________

Urschel CC Carrot Shredder

Frequency

: Nightly

Safety Precautions:

1. Always wear goggles or a full-face shield whenever handling, cleaning, and/or sanitizingproducts.

2. Ensure the equipment is locked at a zero mechanical state prior to beginning work or cleaning.Unplug any electrical service cords.

3. Follow chemical label instructions. Do not mix chemicals without appropriate supervisorauthorization.

4. Wear goggles when using compressed air.5. Wear a wet suit (rain slicker), rubber boots, a plastic hard-hat, and chemical resistant gloves.6. Place plastic bags over electrical motors, electrical boxes, connections, etc. Remove the bags

after the work is completed.

Required Chemicals:

Type Formulation

FCC-2 (Chlorinated Alkaline Cleaner) 1 liter FCC-2 to 10 gallons waterSpecial Acid Cleaner 1 part acid to 5–10 parts waterBio-Guard (Quaternary Ammonia) 200 ppm

Night Procedure:

1. Remove wheel-knife cover guards and wheel-knife assemblies and place in cleaning receptacle.2. Remove and replace any knives requiring sharpening. 3. Remove accumulated produce residues using warm water.4. Apply cleaner to interior and exterior surfaces. Rinse thoroughly.5. Sanitize interior and exterior surfaces.6. Remove accumulated residue, soap, rinse and sanitize interior and exterior framework of

shredder.7. Reassemble all equipment. 8. Inspect for cleanliness.

Biweekly Procedure:

1. Remove wheel-knife cover guards and wheel-knife assemblies and place in cleaning receptacle.2. Remove and replace knives requiring sharpening.3. Remove accumulated produce residues using warm water.4. Apply a thin coat of acid to the interior above-mentioned parts and the interior of the shredder

to remove film build-up; allow applied acid to stand for 8–10 minutes.5. Rinse all parts thoroughly.

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TABLE 4.2Sanitation Standard Operating Procedure (SSOP) for an Urschel Machine (Continued)

6. Sanitize interior and exterior parts of the shredder, including framework.7. Re-assemble all equipment.8. Inspect for cleanliness.

Note:

Brushes or any other cleaning utensil used to help clean the Urschel CC Carrot Shreddermust be identified for this use and stored separately from brushes or any other cleaning utensilsused to clean the floors, bathrooms, etc.

Responsibility:

_______________________

Approval:

_________________________

Source:

Reprinted with Permission from Zagory and Hurst, 1996,

Food Safety Guidelines for theFresh-cut Produce Industry,

IFPA.

TABLE 4.3Operational Standard Operating Procedure for Hand Washing

Date___________

Fresh-cut, Inc.

Page 1 of 1Standard Operating Procedure

___________ Operational SOP #3 Revision #________________

Employee Hand Washing

Purpose:

To ensure proper hand washing and sanitizing to reduce and control the possibility of cross-contamination of product.

Responsible Individual:

Line Supervisor

Procedure

1. All employees shall use anti-microbial soap each time hands are washed.2. Use warmest water possible when washing hands.3. Wash by rubbing hands vigorously together for 20 seconds during each wash. (Do not forget

underneath fingernails and between fingers.) 4. Wash hands at each of the following times:

a. before starting shift each dayb. after each break or when returning to work areac. after handling any non-food item.

5. After thorough rinsing, dip hands 8–10 seconds in an appropriate sanitizing solution beforereturning to workstation.

Corrective Action:

Add sanitizer as needed, replenish fresh sanitizing solution every 4 hours.

Verification:

Quality Control Technician test sanitizer each hour.

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S

ANITATION

A

UDIT

Visual Inspection

Prior to production start-up, line supervisors and the plant sanitarian should visuallyinspect processing equipment to insure that proper sanitation has been completed(Linjacki, 1996). Special attention should be given to observing any grooves,crevices, or inaccessible niches where produce debris has become embedded. Asanitation checklist or log should be used for each production line. Seriously frayedor cracked belts, excess grease accumulation, or loose nuts, hinges, or bolts shouldbe reported to maintenance personnel for correction before the line starts up.Production should begin only after a pre-op inspection has been completed withsatisfactory results.

Microbiological Monitoring

A microbiological survey can be used to evaluate the efficiency of sanitation pro-cedures, ascertain the microbiological loads on equipment in relation to difficultyof equipment cleaning (based on design) and frequency of cleaning, identify areasof contamination throughout the plant that contribute to reduced product shelf lifeand possible pathogen contamination, and verify the HACCP plan (Plusquellec andLeVeau, 1995). Critical areas to test are those that contact produce on a consistentbasis. Microbiological surveys can identify hard-to-reach sites in the plant or onprocessing equipment, which serve as niches for microbiological growth. Wheneveran area is found to be unsanitary (based on high microbiological counts), the samearea should be rechecked to determine if the changes instituted by the sanitationcrew were effective. Data obtained over time from the survey will enable a processorto develop a baseline for the frequency of sampling. These areas should be testedrandomly.

To augment control of sanitary monitoring, many fresh-cut plants have begunusing ATP-bioluminescence. This technology provides virtually instantaneous resultsof the cleanliness of a surface based on the relative light units (RLUs), which equateto the general level of organic residue found (Flickinger, 1997). With ATP kits,processors can make on-the-spot decisions as to whether a piece of equipment orarea has been properly cleaned so appropriate corrective actions can be taken beforestart-up. It is important to recognize that while the ATP data relates the degree oforganic matter present, it is not adequate in judging the microbiological safety ofthe surface (Kornacki, 1999).

Tools to Use

Several microbiological techniques can be used to determine contamination on equip-ment surfaces and, therefore, validate the effectiveness of sanitation. These can bedivided into three categories: traditional, modified, and rapid, real-time assays. Somerepresentatives of each group are listed in Table 4.4. Giese (1995) has published amore complete listing of rapid microbiological test kits and instruments.

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TABLE 4.4Microbiological Procedures for Verifying Sanitation in HACCP Program

Name Method General Procedure Advantages Disadvantages

RODAC (Replicate Organism Direct Agar Contact)

Traditional Pre-poured raised agar (nonselective). Surface is pressed against equipment surface, and plates are incubated 48 hours at 32ºC (89.6ºF) to grow colonies.

FDA/USDA approved. Can vary agar medium. Prepared plates.

Labor intensive to prepare. Can use only on flat, lightly contaminated surfaces.

Swab contact

Traditional Brush sterile, moistened swab over defined area and add sterile water/broth. Prepare appropriate dilutions and add to petri plate. Overlay with sterile agar and incubate 48 hours at 32ºC (89.6ºF) to grow colonies.

FDA/USDA approved. Can use for heavily contaminated areas. Suitable for hard-to-reach areas that are irregular, rough and seamed.

Labor intensive to prepare.

Petrifilm Modified Activated thin film of agar pressed against equipment surface. After rejoining to grid, Petrifilm is incubated 24 hours at 32ºC (89.6ºF) to grow colonies.

Eliminates agar-poured plates. Flexible for use on irregular surfaces. Convenient. AOAC approved for some foods.

Moderate cost.

Redigel Modified Presterilized nutrients in a tube are poured into a petri dish coated with calcium causing a hardened surface to form. Then use like RODAC plates.

Eliminates poured agar plates. Tube does not need heating before adding to petri plates. AOAC approved.

Moderate cost. Not flexible.

Lightning Biolumine-scence System

Rapid, real-time

Uses ATP bioluminescence preparation to determine gross contamination on equipment surfaces within 1–2 minutes.

Portable. No reagent preparation because all chemicals are contained within swabbing unit in ampoules.

Initial investment in luminometer is expensive. Measures total ATP. Cannot detect difference between microbial and nonmicrobial ATP.

(

continued

)

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P

EST

C

ONTROL

A pest-control program is essential to good plant sanitation and must be maintainedby a

certified

pest-control operator (Katsuyama, 1993).

1. Sanitation personnel should maintain an up-to-date inspection of all baitstations, mechanical traps, and glue-stations and should advise pest-controlspecialists of areas within the plant where pest problems are occurring.Bait stations should not be used inside the processing facility. Bait trapsmay be used outdoors.

2. All exterior windows, doors, and building surfaces should be inspectedperiodically to reduce the risk of pests entering the facility.

3. Written records should be maintained by sanitation personnel that includeall of the following: bait station, mechanical trap and glue-station locationsand activity, insect-electrocuting system inspection and cleaning, facilityinspections, and pesticide applications.

4. If an outside pest-control company is hired to manage the program, thein-house quality assurance department should periodically evaluate theeffectiveness of the pest-control program.

EMPLOYEE HYGIENE PRACTICES

The importance of employee personal hygiene to insure food safety has been thesubject of several reviews including those by Troller (1993), Zagory and Hurst (1996)and Marriott (1999). In terms of safety, employees are one of the greatest potentialsources of human pathogen transfer to fresh-cut produce. This is because theymust physically handle the product during its preparation. Salmonella (Pether and

TABLE 4.4Microbiological Procedures for Verifying Sanitation in HACCP Program (Continued)

Name Method General Procedure Advantages DisadvantagesLumac Hygiene Monitoring Kit

Rapid, real-time

Uses ATP bioluminescence preparation to determine gross contamination on equipment surfaces within 1–2 minutes.

Portable. Can determine total ATP or microbial ATP on surfaces. Because microbial ATP can be measured, serves as better indicator of microbial numbers on surface.

Initial investment in luminometer is expensive. Involves more reagent mixing which increases risk of operator error.

Source: Reprinted with permission from Zagory and Hurst, 1996, Food Safety Guidelines for the Fresh-cut Produce Industry, IFPA.

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Gilbert, 1971), Shigella (Davis et al., 1988), and Listeria spp. (Kerr et al., 1993)have all demonstrated the ability to remain viable long enough on food workers’ handsto contaminate the product.

An effective hand-washing regimen is implicit in good hygiene. The debate ofwhether or not to use gloves as a physical barrier to pathogen transfer between thefood handler and food, as well as the effectiveness of hand sanitizers, remains unclear.The 1999 Food Code requires glove compliance by food workers to prevent pathogentransfer to ready-to-eat foods. However, skin can become heavily contaminated in thewarm, moist (due to perspiration) environment under gloves. If the glove is breached,by sharp fingernails or jewelry, then massive contamination through leakage will betransferred to the product (Troller, 1993). While medical literature and regulatoryagencies advocate glove protection, Fendler et al. (1998) reported that the mosteffective hand-washing system was when bare hands were washed and sanitizedhourly. This procedure gave significantly higher hand sanitization levels then whengloves were employed. To minimize skin contamination, Taylor (2000) suggestedusing a protective, antiseptic lotion on hands under gloves. Miller et al. (1994) foundthat instant hand sanitizers resulted in a significant increase in bacterial numbers onhands, while Taylor (2000) reported them useful when washing was not possible,but, they did not have a lasting effect.

Regardless of the method used, employee training in sanitation must begin atthe time of employment. Workers should be given a copy of the company’s rulesfor hygienic practices and dress code when initially hired. Furthermore, managementmust be responsible for developing education programs to instill sanitary awarenessamong employees on a continuing basis.

DEVELOPING AN HACCP PROGRAM

HACCP design, implementation, and maintenance is not easy. It requires strongsupport from top management. A commitment of human and monetary resources isneeded to make the system work. When Pillsbury first decided to implement HACCP,the CEO publicly stated that all raises, promotions, and evaluations would be basedon developing and implementing HACCP to insure safe food production (Stier andBlumenthal, 1995). Now that was a strong statement of support! Employees ultimatelydetermine the success or failure of HACCP. Therefore, training programs are essen-tial to develop a positive attitude about food safety and to help empower personnelto maintain the HACCP program. Because prerequisite programs build the founda-tion of HACCP, these must be reviewed, and their soundness must be verified beforestarting HACCP. Implementing HACCP takes time. Experience has shown thatinstallation and implementation takes between six months and two years.

ASSEMBLE THE HACCP TEAM

Once management agrees to the HACCP concept, an HACCP coordinator shouldbe appointed to lead, coordinate, and build the HACCP team. The team leader mayrequire an education in HACCP. In addition to leadership and coordination skills,this person should be creative, have good communication and listening skills, and

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64 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

have an overall knowledge of the plant operation and its products. A qualified teamleader is crucial to initiating the HACCP program and insuring it will be implementedand maintained in the correct fashion.

Because HACCP is a systems approach to safety, the team must have multital-ented personnel from both management and production involved. They should havea real working knowledge of the operation and products. Fresh-cut produce teamsshould consist of managers, quality assurance/sanitation personnel, production oper-ations, engineering/maintenance, purchasing/procurement, marketing, and labor (on-line personnel). The leader must instruct the team on HACCP and what the role ofthe team will be.

HACCP TRAINING

Because the level of technical expertise needed by the HACCP team will obviouslybe greater than that needed for the rest of the employees, this training is best carriedout in two phases (Mayes, 1994).

• Phase I: Training for managers, supervisors, and HACCP team membersshould focus on hazard analysis and risk assessment issues, drawing flow-charts, determining critical control points in the process, and collectingdata on control charts. This training consists of seminars, workshops, andeven one-on-one instruction of how to design, implement, and maintainHACCP procedures.

• Phase II: Training for front-line production, maintenance, and sanitationwill most often involve changing attitudes concerning handling activities,creating employee awareness, and paying attention to detail, as it relatesto safety. Line employees must be taught the difference between qualityconcerns and safety concerns. Most importantly, employees need to under-stand that the HACCP might change their specific job activity and whythis change is needed.

In staff training, keep in mind the ethnic and cultural differences of employees.English is not the primary language for a significant number of fresh-cut produceworkers. Often out of fear of being incompetent or of losing one’s job, an employee willindicate an understanding of policies or procedures when this might not be the case.Bridge the communication barrier and assure comprehension by providing training andoperating manuals in the native language or languages of your employees (Rosete, 1998).

DEFINE THE PRODUCT

HACCP must be custom-designed for each fresh-cut product in a given plant oper-ation. Although generic models can serve as useful tools to demonstrate how tocreate an HACCP plan, these models are not always applicable for every product,processing line, or specific plant facility. It is important to realize that HACCP is nota turnkey system. Each HACCP plan will be product, processing line, and plant specific.

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Before HACCP design begins, several preliminary tasks must be accomplished.Once the HACCP team has been assembled and trained, each product must be described,and its intended use must be defined. This consists of detailing its form, size, packagingand storage requirements, method of handling, and intended customer. Table 4.5shows a sample product description, distribution, intended use, and customer.

DEVELOP A FLOW DIAGRAM

A flow process should be constructed for each product to detail how the product iscreated from raw material production, through processing and packaging, to distri-bution and consumer use. Its purpose is to identify specific areas where hazardscould occur. Although a complete flow diagram takes into consideration the entireproduction process from farm to table, the HACCP plan for a processor usually involvesonly those parts of the process where control can be exercised (see Figure 4.4).Fresh-cut processors use vendor verification programs to document the safety oftheir raw ingredients before processing and retail/wholesale audits to verify safehandling after leaving the production plant. Once completed, the flow diagram shouldbe verified with on-line supervisors for completeness and accuracy. Often, it must bereformatted or revised using descriptive words that are more easily understood byall members of the HACCP team. The flow diagrams should be reviewed on aquarterly basis. A number of computerized software programs (e.g., doHACCPZversion 2.3, and HACCP Documentation Software, version 2) have made updatingflow processing changes to an operation quick and easy.

TABLE 4.5Product Description, Use, and Distribution

PRODUCT DESCRIPTIONCommon Name:

Shredded Lettuce; prepared from refrigerated iceberg lettuce; trimmed, cored and cut; washed in a solution of potable water and chlorine

Type of Package:Packed in food grade plastic bags, 8 oz. to 10 lb. units

Length of shelf life, at what temperature?:Optimum shelf life of 14 days if refrigerated at 34° to 38°F (1.1° to 3.3°C)

Labeling Instructions:Bag and/or box contains “processed on” or “use by” date

Where will it be sold?:Foodservice operations and retail markets

Intended use and consumer:For use in salads and sandwiches for foodservice customers; prepackaged units for in-house use by consumer

Special Distribution Control:Product distributed under refrigeration, stored in refrigeration at 34° to 38°F (1.1° to 3.3°C)

Source: Reprinted with permission from IFPA Technical Committee, 2000, HACCP for the Fresh-cutProduce Industry, IFPA.

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66 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

PERFORM A HAZARD ANALYSIS (PRINCIPLE 1)

The first step in formal HACCP design is to conduct a hazard analysis (Principle 1)of the process. The purpose of hazard analysis is to list all potential hazards that aresignificant enough to likely cause illness or injury if not effectively controlled. Anextensive listing of possible hazards of a microbiological, chemical, or physicalnature that might be imparted to food has been reviewed by Rhodehamel (1992).

FIGURE 4.4 Example of a fresh-cut lettuce operation.

Process Flow Diagram

Raw lettucereceipt/inspection

Raw storage cooler

Automatedfilling/weighing machine

Spin

Spinner basket

Water flume wash

Size reduction (chopping,dicing, slicing)

Product preparation(cutting, coring, grading)

Finished cold storage BoxPalletize

Heat seal Metal detector

Dry storage

Packaging material receipt

Distribute

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Hazard analysis is a two-step procedure. First, the HACCP team must identify thepotential of all hazards occurring at each step in the flow diagram. Once identified,it is important to consider how the hazard may become incorporated into a fresh-cutproduct. Brainstorming and cause-and-effect analysis are two quality improvementtechniques that can be used to assess sources of hazard contamination (Rao et al.,1996). Brainstorming is a technique used to generate, through divergent thinking, alarge number of creative possibilities to the cause of a problem. A cause-and-effectanalysis can provide additional structure to brainstorming by grouping the conver-gence of ideas generated into key causes that will be addressed by further action.

Using a cause-and-effect diagram, the relationship between a given effect orproblem and all identified causes of that effect can be narrowly focused. The effector problem (e.g., microbiological contamination) is represented by a horizontal arrowor spine, and principal causes are identified by arrows entering the spine. Principalcauses of any effect are inherently found in six major sources of variability, namely,machines, materials, methods, measurement, personnel, and the environment, all ofwhich are part of any process. Each principal arrow can have secondary arrowsrepresenting sub-causes. Figure 4.5 illustrates part of a cause-and-effect diagramhighlighting the environment as a possible source of Listeria contamination to fresh-cut lettuce. Once a comprehensive cause-and-effect diagram has been constructedand all possible causes of a problem verified, appropriate measures must be institutedto control the hazard.

Step two of the hazard analyses is an evaluation of each identified hazard basedon its risk and severity. Risk is an estimate of the likely occurrence of a hazard, whileseverity is the seriousness of the hazard. For example, Listeria monocytogenes andClostridium botulinium are potential microbiological hazards in the fresh-cut industry.

Epidemiological data and scientific research, however, have shown L. monocy-togenes to be a greater risk in fresh-cut products, even though C. botulinium wouldbe considered the more severe hazard based on mortality rates. Thus, routine screen-ing of the processing environment for L. monocytogenes would be warranted to keepit out of fresh-cut products.

After all potential hazards have been listed, and control measures have beenagreed upon, the HACCP team must face the challenge of determining the “signif-icance” of each identified hazard based on risk assessment methodology. Tradition-ally, hazard analysis has been evaluated using qualitative risk assessment proceduresthat are subjective and do not adequately validate product safety (WHO, 1995;Buchanan, 1995). One method of gaining quantitative information is through theuse of predictive microbiology, where bacterial growth responses are summarizedunder different environments to form mathematical equations (McMeekin et al.,1993). In recent years, several authors (Notermans et al., 1995a; Miles and Ross,1999) have published research demonstrating “quantitative microbiological riskassessment,” where predictive models have been integrated into HACCP plans toprovide a more objective way to quantify and rank microbiological hazards basedon risk assessment. Because present predictive microbiology instruments, microbi-ological challenge testing (MCT), and storage testing (ST) are slow and expensive,Panisello and Quantick (1998) have developed computerized predictive microbiol-ogy software to assist in microbiological risk assessment in the HACCP system.

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A thorough hazard analysis is essential to the design of an effective HACCPplan. If this is not done correctly and the significant hazards warranting controlwithin the plan are not properly identified and evaluated, then HACCP will not beeffective regardless of how well the plan is followed (Bernard, 1997). As shown inTable 4.6, when the HACCP team does a hazard analysis in the production of fresh-cut lettuce, microbiological pathogens would be the biological hazard associated

FIGURE 4.5 Part of a cause-and-effect diagram highlighting the environmental componentas a source of L. monocytogenes contamination.

Cause-and-Effect Diagram

Poor sanitation inproduce cooler

Poor sanitation inprocessing room

Personnel

Measurements Methods Materials

Listeria-contaminatedlettuce

EnvironmentMachines

Floor (waterpuddles)

Cleaning aids(brushes, sponges)

Drains (water intrench/gutter)

Refrigerationunits

Case

Drippans

Fan blades

Condensate

AirHoldingracks

BinsPallets

Electricalconduits

Ceiling

Overheadpipes

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with the water flume wash step. Pathogen buildup would be significant, if not con-trolled, because of water reuse and cross-contamination over time. Proper chlorina-tion is the control measure applied to reduce this hazard.

IDENTIFY CRITICAL CONTROL POINTS (PRINCIPLE 2)

Once the significant hazards and control measures have been identified throughhazard analysis, the HACCP team must determine the best place to control thesehazards in the process. This process, called establishing the critical control points(CCPs), is the heart of the HACCP plan. A critical control point is a step at whichcontrol can be applied and is essential to prevent or eliminate a food safety hazardor reduce it to an acceptable level (NACMCF, 1998). For every significant hazardidentified, one or more CCPs must be designated to control the hazard. The CCPsare the points in the process where HACCP control activities will occur. But, it maynot be possible to fully prevent or eliminate a significant hazard, only minimize itto an acceptable level. For example, chlorination of flume water in the fresh-cutindustry is practiced to minimize the introduction of pathogens into the water throughcross-contamination of the produce items being washed. Using chlorinated water torinse sliced chicory leaves for “ready-to-use” salads, Nguyen-The and Prunier (1989)found that, on the average, 103 bacteria per milliliter were inoculated into the washwater. This is low compared to the numbers 105 to 106 cfu per gram found on theproduct, but it demonstrates that microorganisms can be rinsed off fresh-cut surfacesby washing. Although no human pathogens were reported, presumably, they mayhave been part of the microflora. It is known that chlorine efficacy as a sanitizer is

TABLE 4.6 Excerpt from Hazard Analysis Worksheet

HAZARD ANALYSIS PROCEDURE (Principle 1)

Process Step

Biological Chemical Physical

Potential Hazard

Controlled, Introduced, or Enhanced

Is Potential Hazard

Significant? Yes/No

Justification for

Determination of Hazard

Significance

What Control Measures Can Be

Applied to Reduce or

Eliminate this Hazard?

Water flume wash

B Pathogen controlled

Y Water reuse; pathogen introduction into water through cross-contamination

Chlorine added to and monitored in wash water; water cold and has optimum pH for chlorine action

Source: Reprinted with Permission from IFPA Technical Committee, 2000, HACCP for the Fresh-cutProduce Industry, IFPA.

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70 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

affected by certain physiochemical characteristics of water. Chlorine antimicrobialactivity against both plant and human pathogens (Bartz and Eckert, 1987; Maier etal., 2000) decreases as the particulate matter in the water increases. Particulate mattermay interfere by either chemically reacting with the disinfectant, thus neutralizingits action, or physically shielding the organism from the disinfectant. Clearly, thegoal in chlorinating flume water is to maintain levels at ≥1 ppm free availablechlorine (IFPA, 2000) and to keep any pathogens from reaching infectious levels inthe water which may be imparted to fresh-cut products.

Many points in a flow diagram not identified as CCPs may be considered controlpoints. A control point (CP) is any step at which biological, physical, or chemicalfactors can be controlled (NACMCF, 1998). Many types of CPs can exist in a fresh-cut operation. They include those that address quality control (color, flavor, texture),sanitary control (SSOPs, GMPs), and process control (filled weights, seal closures).Pinpointing the right CCPs is a most crucial and problematic aspect of an effectiveHACCP program (Demetrakakes, 1997). HACCP is about safety, not about quality.Perhaps the biggest mistake processors make is to define too many CCPs, some ofwhich are really CPs, in their HACCP program. Quality and safety issues areconfused for two reasons. Quality is vital to a product’s well being in the marketplace,which can tempt processors to elevate it to the highest level of scrutiny. In addition,most processes have points where a breakdown could affect both quality and safety.

To keep HACCP programs plant-friendly and sustainable, Bernard (1997) rec-ommends that the number of CCPs should be kept to a minimum and none shouldbe redundant. Redundancy will add to the cost of record keeping. Experience hasshown that HACCP plans that are unnecessarily cumbersome will likely be the onesthat fail. A CCP should be limited to that point or those points at which control ofthe significant hazards can best be achieved. For example, a metal hazard can becontrolled by ingredient sourcing, magnets in the water flume, screens, and a metaldetector, all in one processing line. However, sourcing magnets and screens wouldnot be considered CCPs (but instead upstream CPs). A possible metal hazard is bestcontrolled by use of a metal detector and product rejection at the end of the packagingline (Lockwood et al., 1998). This is an example of where the CCP can be severalprocess steps away from the point where a significant hazard may be introduced.

To assist in finding where the correct CCPs should be located in a unit operation,we can use a tool called the CCP Decision Tree. Several versions of this tool canbe found in the literature (NACMCF, 1998; Mortimore and Wallace, 1994; Wedding,1999a). The decision tree is a logical series of questions that are asked for eachidentified hazard at each process step. The answer to each question will follow aprocess of elimination and ultimately lead to a decision as to whether or not a CCPis required at that step. Using a CCP decision tree promotes structured thinking andinsures a consistent approach of every hazard at each step. It also has the benefit offorcing and facilitating HACCP team discussion, teamwork, and study (Mortimoreand Wallace, 1994). However, as pointed out by Wedding (1999a), this is not aperfect tool and is not a substitute for common sense and process knowledge, becausecomplete reliance on the decision tree may lead to false conclusions. Application ofhow to use the CCP decision tree for metal contamination at the water flume isdemonstrated in Figure 4.6. If the answer to Q1, as shown, is yes, then ask Q2. To

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Safety Asp

ects of Fresh

-cut Fru

its and

Vegetab

les71FIGURE 4.6 Using CCP decision tree to illustrate whether metal contamination at the water flume step should be a CP or a CCP.

Application of CCP Decision Tree

Q1: Do control measures existfor the identified hazard?

Modify step, process, or product

Is control of this step necessary forsafety?

Yes

Q2: Does this step eliminate or reducethe likely occurrence of a hazard toan acceptable level?

Q3: Could contamination with identifiedhazard(s) occur in excess of acceptable level(s),or could these increase to unacceptable level?

Q4: Will a subsequent step, prior toconsuming the food, eliminate theidentified hazard(s) or reduce the likelyoccurrence to an acceptable level?

Step: Water flume

Identified Hazard:Physical (metal contamination)

Control Measures:In-line magnet

NOYes

Yes

NO

Yes

CCP

NO

STOP

NOT ACCP

STOP

NOT A CCP

NO

CCP

NO

NOT A CCP

STOP

Yes

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answer this question, consider if this is the best step at which to control the hazard.If the answer is yes, then the step is a CCP; move to the next step with a significanthazard. If the answer is no, as shown, then ask Q3. This question refers to contam-ination occurring or increasing to unacceptable levels beyond this step. If the answeris no, then the step is not a CCP for that hazard. If the answer is yes, then ask Q4. Ifthe answer is no, then the step is a CCP. If the answer is yes, then this step is not a CCPfor the hazard. In this case, magnets are a CP, but not a CCP because metal contami-nation can occur anywhere in the processing line. The best location to designate aCCP for metal contamination in the product is at the end of the packaging line.

HACCP teams often ask, “How many CCPs do we need in our HACCP plan?”(Wedding, 1999a). Unfortunately, there is no simple, clear-cut answer to this ques-tion. It depends on plant layout and design, product being produced, ingredientsused, equipment age and condition, processing methods employed, and, especially,the effectiveness of the prerequisite programs implemented. The situation often ariseswhere a similar step may be designated a CCP in one facility but covered under aprerequisite program in another facility. Each processor must determine the bestlocation to designate CCPs in his unique operation (IFPA, 2000).

SET CRITICAL LIMITS (PRINCIPLE 3)

Critical limits are the safety boundaries that must be established for each identifiedCCP in the hazard analysis step. A critical limit (CL) is a maximum and/or minimumvalue to which a biological, chemical, or physical parameter must be controlled ata CCP to prevent, eliminate, or reduce to an acceptable level the occurrence of afood safety hazard (NACMCF, 1998). It must be understood that CCPs do not imple-ment the control of hazards; they are just affected by them. The parameters thatactually control hazards are the critical limits. They are individual values, notaverages, that signify whether the control measure at a CCP is “in” or “out” of control(Wedding, 1999b). Critical limits define the boundaries between safe and unsafeproduct. For the HACCP team to set correct critical limits, they must have in-depthknowledge of the potential hazards, full understanding of the factors involved intheir prevention or control, and knowledge of the control mechanisms of the process(Mortimore and Wallace, 1994). Criteria or factors that make up critical limits canbe grouped into several categories as demonstrated in Table 4.7.

Because critical limits must be measurable in real-time, microbiological limitsare not suitable for controlling CCPs. Conventional testing takes days for results,

TABLE 4.7Criteria Utilized in the Fresh-cut Industry to Set Critical Limits

Chemical PhysicalChlorine TemperaturepH Absence of foreign materials (metal, etc.)Titratable acidity ORP (millivolts)

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and even rapid screening methods can take several hours. Microbiological limitswill not allow instant action to be taken when a CCP deviation occurs. Also, theprobability of low pathogen levels, nonrandomly distributed through a batch, sig-nificantly increases the chance of failing to detect pathogens when statistical sam-pling is used (Moberg, 1992).

A recent exception to the general rule of microbiological ineffectiveness hasbeen the development of ATP bioluminescence. Although truly a rapid testingmethod, i.e., results in minutes rather than hours, it is used by some fresh-cutprocessors to verify hygiene control at CCPs. But, this new technology has severallimitations. ATP is nonspecific in distinguishing between eukaryotic and microbio-logical ATP (Griffiths, 1996), its light signal may be quenched or enhanced bycleaning/sanitizing residues (Velazquez and Feirtag, 1997), and the limit of its sensi-tivity is 1000 cells (Forsythe and Hayes, 1998), which is greater than the infectiousdose for several food pathogens, including E. coli O157:H7, Listeria monocytogenes,and Shigella spp. (Doores, 1999). These limitations make ATP a better tool for mon-itoring hygienic compliance with prerequisite programs (e.g., SSOPs and GMPs)than serving as a critical limit parameter.

When a critical limit is violated, it signals that a potential hazard may be introducedat that CCP, thus, immediate control measures must be taken to bring the CCP backwithin its CL range. Exceeding the control limit indicates that one of the followingsituations has occurred (Moberg, 1992):

• evidence of the existence of a direct health hazard (e.g., detection of Listeriamonocytogenes in a fresh-cut salad product)

• evidence that a direct health hazard could develop (e.g., no chlorine orother antimicrobial agent used to sanitize recirculated flume wash water)

• indications that a product was not produced under conditions assuringsafety (e.g., metal detector not running during a production shift)

• indication that a raw material may affect the safety of the product (e.g.,E. coli O157:H7 found in whole cabbage heads from supplier)

Each CCP must have one or more CLs set for each significant hazard, and thesemust be scientifically based (NACMCF, 1998). In many cases, the appropriate CLmay not be readily apparent or available to HACCP team members. Wedding (1999b)has listed some sources to consult for his information. These include research articles,government documents, trade association guidelines, in-plant studies, universityextension publications, and industry experts. If outside sources are used to establishCL, they should be documented and become part of the HACCP plan as shown inTable 4.8.

Because of the need for “real-time” monitoring and quick data feedback, chemicaland physical measurements made at CLs also serve as an indirect measure of micro-biological control at the CCPs. In these instances, correlation between chemical andphysical parameters and microbiological parameters must be predetermined in orderto set safe control limits. With this correlation, exceeding a chemical or physical limitwould mean that the corresponding microbiological limit had been violated, and apotential health hazard may exist or develop (Moberg, 1992). Notermans et al. (1995b)

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TABLE 4.8Excerpt of HACCP Plan Summary Page for Fresh-cut Lettuce

Process Step CCP #

Biological Chemical Physical

Hazard Description Critical Limits

Monitoring Procedures/Frequency/

PersonResponsible

Corrective Actions/Person

Responsible

Verification Procedures/

PersonResponsible HACCP Records

Water flume wash

CCP 1B

L. monocytogenes

E. coli O157:H7

Salmonella and Shigella spp. and other microbial pathogens

Potable water containing ≥1 ppm free residual chlorine for 30 seconds; at pH ≤7.0

Prior to start of processing and each 30 minutes thereafter, QC personnel will monitor free chlorine using standardized test kit, and a calibrated pH meter will be used to monitor water pH three times per shift

QC personnel will adjust water chemistry to maintain pH and chlorine added to maintain CL; held product will be rewashed and CL deviations noted in a log

QC personnel will maintain chlorine monitoring logs, pH temperature monitoring logs, CCP deviation/ corrective action logs, calibration logs for thermometer, pH test and chlorine test kit used, microbiological tests will be run on finished product at least once/year to validate pathogen absence, HACCP plan will be revalidated at least once/year

HACCP coordinator reviews all HACCP records weekly, HACCP coordinator will conduct calibration tests, plant manager reviews records daily, state food inspector/ FDA audits, customer audits/ internal/consultant audit once/year, all records kept at least one year, random sampling/ testing product to ensure process verification

Source: Reprinted with Permission from IFPA Technical Committee, 2000, HACCP for the Fresh-cut Produce Industry, IFPA.

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proposed a method to quantify microbiological levels and their reduction at eachCCP. This approach would require knowledge of the kind and number of potentiallyhazardous microorganisms in the raw food material and the use of computer models,storage tests, and microbiological challenge testing to predict microbiological survivaland death rates at each stage of the production process.

Once CLs have been set for all CCPs, the task is to keep the parameter beingmeasured in control within the established tolerances. This may or may not be aneasy job depending on the kind of variation in the process. Establishing operatinglimits is a practical means to help prevent routine violation of the CLs (Wedding,1999b). Operating limits are criteria that are more stringent than critical limits andare established at a level that would be reached before the critical limit is violated(Lockwood et al., 1998). Process adjustment should be taken when the operatinglimit is exceeded to avoid loss of control and the need to take corrective action atthe critical limit.

ESTABLISH MONITORING PROCEDURES (PRINCIPLE 4)

Monitoring is one of the most important components of the HACCP plan, becauseit is what the HACCP team relies upon to maintain control at the CCPs. It documentsthat a process can operate consistently within the CL to control the identified hazards.By definition (NACMCF, 1998), monitoring a planned sequence of observations ormeasurements to assess whether a CCP is under control produces an accurate recordfor future use in verification. Monitoring serves three purposes: first, to track theoperation of a process and enable the identification of trends toward a critical limitthat may trigger process adjustment; second, to identify when and where there wasa loss of control (a deviation occurred at the CCP) such that corrective action isneeded; and third, to provide written documentation of the process control system(Lockwood et al., 1998).

The HACCP team will be responsible for designing the monitoring activities ateach CCP. Procedures must identify what control measures will be monitored, howfrequently monitoring should be performed, what procedures will be used (data col-lection methods and equipment), and who will perform the monitoring.

WHAT WILL BE MONITORED?

Monitoring usually means measuring a physical, chemical, or sensory parameter, ortaking an observation of the product or process to determine compliance within aCL. Examples in the fresh-cut industry include the following:

• checking that a vendor’s certificate for safety accompanies a lot of producematerials (required by some customers)

• measuring free chlorine levels in the flume wash water• checking the metal detector at the end of the packaging line• measuring packaged product temperatures at the end of the processing

line (required by some customers)

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WHEN WILL MONITORING BE PERFORMED?

To assume adequate control, the frequency of monitoring must be consistent withthe needs of the operation in relation to the variation inherent in the control step(Bernard, 1997). Continuous monitoring at a CCP is always preferred, but some-times, it may not be practical or available for use. When it is not possible to monitora CL at the 100% level, or continuously, then it is necessary to establish a monitoringinterval (e.g., discontinuous) that will be reliable enough to insure the hazard inquestion is under control. It is vital that the sampling procedures used to monitorCCPs be statistically valid. Data collection programs will be most accurate and ofgreatest benefit if established under a structured system of statistical process control.An example of a discontinuous monitoring system (e.g., measuring free residualchlorine at specified intervals) for the CCP designated at the water flume wash stepof fresh-cut lettuce is described in Table 4.8.

HOW WILL MONITORING BE PERFORMED?

As noted earlier, physical and chemical measurements are the preferred monitoringmethods, because testing can be done rapidly. The equipment chosen for CCP moni-toring must have the degree of sensitivity to accurately control hazards. In fresh-cutprocessing, CCP monitoring equipment includes thermometers, pH meters, ORPmeters, chlorine test kits and probes, metal detection units, etc. Daily calibration orstandardization of this equipment is necessary to insure accuracy. Records shouldbe maintained on the equipment calibration and must become part of the supportdocumentation for the HACCP plan.

WHO WILL PERFORM THE MONITORING?

An underlying concept of HACCP is to promote safety awareness to production lineemployees. A good way to achieve this goal is to involve them in the monitoringactivities. Monitoring by the personnel and equipment operators can be advantageousbecause they are continuously viewing the product and/or equipment and are in thebest position to observe changes from the norm quickly. Also, there is the benefitof a broad base of understanding, commitment, and ownership to the HACCP program.The role of quality control personnel may be more appropriate in verification or“checking the checker” (Bernard, 1997).

According to Lockwood et al. (1998), those responsible for monitoring a controlmeasure at a CCP must do the following:

• be trained in the CCP monitoring techniques• fully understand the importance of CCP monitoring• have ready access to the monitoring activity• accurately report each monitoring activity• immediately report critical-limit infractions so that immediate corrective

actions (Principle 5) can be taken

Should any unusual occurrences and/or deviations from the CL occur at a CCP,the monitor is responsible for taking corrective action immediately. All process

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adjustments must be reported on the corrective action form. All records and docu-ments associated with CCP monitoring must be signed or initialed by the persondoing the monitoring.

STATISTICAL MONITORING FOR PROCESS CONTROL

An underutilized tool for monitoring and verifying HACCP systems is the use ofStatistical Process Control (SPC). Implementation of SPC techniques is the mosteffective way to achieve process control. What is process control? Hubbard (1996)defines it as a process functioning or operating within its optimum level of capabilitywith only common cause (inherent) variation occurring among its manufacturedproducts. The kind of variation and its capability of being controlled can only bedetermined by the application of applied statistics. Hurst (1996) presented a systematicguide for implementing statistical tools in the monitoring of quality of fresh-cutproducts that could also be applied to safety issues.

Process control is not a natural state. Most processes do not operate in a stateof control. Furthermore, once in control, a process will not remain there. It is notknown whether a process is “in” or “out” of control until it is measured using SPCtechniques. SPC is a quality management system that uses graphical and statisticaltools to analyze, control, and reduce variation within a process. The heart of SPCis the Statistical Control Chart, the specific tool for monitoring process control.Control chart theory is based upon the notion that the parameter being measured,when in statistical control, will vary normally (e.g., only common cause variation)about a central value (ICMSF, 1988). Control chart methodology is the only SPCtool that can distinguish between common-cause (inherent) and special-cause (unnat-ural) variation in a process. The control chart allows the highlighting of special-cause variation, if present, when monitoring a process. If the special-cause variationsource can be found and eliminated in the process, then the process will exhibit onlycommon-cause variation. Only when common-cause variation is present, is the processin a state of “statistical control.” What makes statistical control so important? Theessence of statistical control is predictability. A process is predictable when it is ina state of statistical control, and it is unpredictable when it is not in a state of statisticalcontrol (Wheeler and Chambers, 1992). In addition to predictability, there are twoother sound reasons for putting a process into statistical control (Clements, 1988).The process becomes stable (meaning it is operating normally with only the expectedamount of variation), and it becomes capable (meaning the parameter being measuredcan meet the specification or boundaries within which it is supposed to operate).The most effective way to avoid a safety hazard is to have a process that is stable,capable, and predictable.

INTEGRATING SPC AND HACCP

Both SPC and HACCP focus on process control. The difference between the two isthat HACCP focuses on safety issues, while SPC has addressed quality and produc-tion control issues. In terms of process control, however, HACCP has two inherentlimitations. First, it has historically been implemented as a management tool for

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product safety, not for production line process control (Giese, 1999). Second, theHACCP monitoring system takes an attribute approach to safety. The process atwhich some parameter, like temperature, is being monitored at the CCP (CriticalControl Point) is either categorized as “safe” (within critical limit) or “unsafe”(exceeding its critical limit). Attribute charting is not a good warning tool for signalingan impending change in the process leading to a safety hazard in the product.Attribute charts do not detail the relative position of a parameter being monitoredwithin its critical limit. Therefore, they are poor tools for anticipating processchanges (Surak et al., 1998).

So, how can HACCP be made a more effective prevention tool for safety hazardsin the production process? The key lies in integrating SPC techniques into theHACCP plan. The focus must be on the process not the product. Also, a new typeof process control chart must be implemented. Specifically, variable control chartsare used. Variable charts are better than attribute charts for monitoring and verifyingprocess control at HACCP CCPs.

Variable charts should be used for quantifying and measuring process outputand identifying if this output can remain within statistically defined control limits.More importantly, variable charts can signal the beginning of trouble in a process basedon the pattern of points plotted so that action can be taken before a safety hazardhas time to become incorporated into the product.

The integration of SPC into an HACCP plan will increase its effectivenessbecause both systems focus on prevention, both are process-related tools (if variablecharts are used), both can be used to document and verify safe product to customers,and both meet regulatory requirements.

SPC MONITORING OF CCPS

Statistical control charting is ideally suited for HACCP monitoring of designatedCCPs, because it provides an early warning signal of when to take corrective actionbefore a CCP exceeds its CL. The application of SPC allows a fresh-cut processorto control his CCPs systematically, predictably, and most importantly, demonstrably(Grigg, 1998). To be able to predict future safety of a product to customers and havethe statistically valid evidence (documented charts) to back up your statements is apowerful marketing tool.

It is important to note, however, that process control may not be HACCP control.If the common-cause variation of the parameter monitoring a CCP is too great, theprocess may exceed the critical limit. Thus, a process in statistical control may notbe capable of producing a safe product. Likewise, the parameter monitoring a CCPmay be within the CL but not in statistical control. In fact, any one of four scenariosmay exist, as demonstrated in Figure 4.7.

There is a major difference in the manner of setting SPC limits vs. HACCP limits.SPC limits are set based on the standard deviation of the statistic plotted from thedata when the state of the process is assumed to be in statistical control. In other words,the width of the SPC control limits are based on the common-cause variation exhibitedby the process. In contrast, HACCP critical limits must be set based on valid researchevidence that has demonstrated boundaries of safety for the statistic being plotted.

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SPC limits must have less variability than HACCP limits (Van Schothorst and Jongeneel,1994).

It must be remembered that any statistical chart that relies on plotted data averagesmay obscure extreme values that could pose a health hazard (ICMSF, 1988). Whileplotted averages for a CCP may be within critical limits, individual values may beabove or below the CL for safety. For this reason, it is recommended to first monitorCCPs using individual values plotted on individual/moving range charts (Surak et al.,1998) to be certain they can remain within their predetermined CLs. Once processstability has been achieved, then one can proceed to construct average/range charts.These are better indicators of any process shift that may occur for a CCP within the CL.

FIGURE 4.7 Chlorine monitoring of wash water using SPC and HACCP methodology.

_

Applying SPC to Monitor HACCP CCPs

Legend: UCL — upper control limit ( )LCL — lower control limit ( )CL — critical limit ( ) X — mean ( )

UCL

Time (0.5 hr)

pp

m c

hlo

rin

e

X

120

80

LCLCL40

Chlorine Monitoring

UCL

Time (0.5 hr)

pp

m c

hlo

rin

e

X

120

80

LCLCL40

Chlorine Monitoring

UCL

Time (0.5 hr)

pp

m c

hlo

rin

e

X

120

80

LCLCL40

Chlorine Monitoring

UCL

Time (0.5 hr)

pp

m c

hlo

rin

e

X

120

80

CLLCL

40

Chlorine Monitoring

(d) Process out of statistical controland outside CL

(c) Process out of statistical controlbut within CL

(b) Process in statistical controlbut outside CL

(a) Process in statistical controland within CL

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ESTABLISH CORRECTIVE ACTIONS (PRINCIPLE 5)

Although HACCP is designed as a preventative strategy to hazards entering theprocess, ideal conditions do not always prevail. There may be deviations from CLs,and corrective actions may be needed. By definition, a corrective action is takenwhen there is a CL deviation identified by monitoring a CCP (NACMCF, 1998).Tompkin (1992) pointed out that corrective action involves four activities:

1. Bring the process back into its CL through process adjustment2. Determine and correct the cause of the deviation3. Determine the disposition of the noncompliant product4. Record the corrective action taken and the disposition of the noncompliant

product

When CLs are violated at a CCP, predetermined (developed in advance for eachCCP and included in HACCP plan) corrective action procedures must be instituted.The goal is to adjust the process on the spot to minimize the amount of noncompliantproduct and determine the safe disposition of the affected product (Lockwood et al.,1998).

The great diversity in fresh-cut products and the variation in equipment, type ofprocessing, raw material quality, etc., require that specific corrective actions be devel-oped for each CCP according to the parameters of the processing. Examples of correc-tive actions to be taken if the CL for chlorine in water fails can be found in Table 4.8.

Individuals who have a thorough understanding of the product, process, andHACCP plan should be assigned responsibility for writing and overseeing that thecorrective actions are implemented (Bernard, 1997). At a minimum, the HACCPplan must specify what is done when a deviation occurs, who is responsible fortaking corrective action, and what happens to the noncompliant product. Detailedaccurate records must document all of these actions and procedures.

ESTABLISH VERIFICATION PROCEDURES(PRINCIPLE 6)

The purpose of the verification step is to confirm through documentation that theHACCP plan is followed as designed and implemented. The verification step pro-vides assurance that the HACCP program is achieving the established objective offood safety (Prince, 1992) and that the plan will operate consistently on a day-to-day basis. Verification is defined as those activities, other than monitoring, thatdetermine the validity of the HACCP plan and determine that the system is operatingaccording to the plan (NACMCF, 1998).

An inherent aspect of verification is the initial validation of the HACCP plan.Before implementing HACCP, the HACCP team must review the plan to determinethat it is accurate in all details. Validation, as defined by Stevenson and Gombas(1999), is that element of verification that focuses on collecting and evaluatingscientific and technical information to determine if the HACCP plan will effectivelycontrol the hazards.

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According to Hudak-Roos (1999), verification is a daily activity that shouldanswer the following question: Are your activities in compliance with the written/implemented HACCP plan? In contrast, validation is a periodic function performedevery six months to a year. It should answer the question: where is the scientificevidence that the HACCP plan has been properly designed? Simply put, validationis the proof that the intended result can be achieved. It is evidence of process capability(Sperber, 1999).

SPC VALIDATION/VERIFICATION OF A PROCESS

For each CCP in the HACCP plan, there will be a need to validate that, under normaloperating conditions, the process can be maintained within its CL. In other words,is the process verifiable? An ideal way of assessing whether a process is capable ofremaining within specified limits is to use statistical analysis. Statistical validationof a process to determine the probability (confidence) of its ability to stay withinspecified (critical) limits is known as establishing its process capability (Mortimoreand Wallace, 1994). Remember, all processes should be validated before the HACCPplan is implemented. Conducting a process capability analysis accomplishes twogoals (see Figure 4.8). First, can the process be validated so that it is capable ofachieving the Critical Limits (CLs) that have been established? Second, can theprocess be verified so that it is capable of being controlled? Can it realisticallyremain within the CLs on a day-to-day basis? A process capability study validateswhether or not CCP control is achievable and verifies whether or not the processcan remain consistent. The statistical format for calculating process capabilities hasbeen reviewed by Hubbard (1996) and is graphically demonstrated in Figure 4.8.

Each HACCP plan will include verification procedures for individual CCPs. Themajor verification activities include plant audits, calibration of instruments/equipment,targeted sampling and microbiological testing, and HACCP records review (Lockwoodet al., 1998).

Audits are conducted by an unbiased person not responsible for performingmonitoring activities. The objective is to compare actual practices with what is writtenin the HACCP plan. Audits can be performed by a member of the HACCP team,plant management, outside experts or consultants, regulatory agencies, and customers.

Calibration of instruments and equipment used to monitor CCPs is extremelyimportant. If the monitoring devices are out of calibration, then results will not beaccurate. Significant deviations might go unnoticed, creating a potential health hazard.If this happens, the CCP could be considered out of control because the last docu-mented acceptable calibration. This situation must be considered when establishingmonitoring frequency.

Verification may also include targeted sampling and microbiological testing. Vendorcompliance can be checked by targeted sampling when receipt of material is a CCPand purchase specifications are relied on as control limits. Microbiological testingcan be used as a verification tool to determine if the overall operation is under control.

All records connected with the above activities must be reviewed. Especiallyimportant are CCP records; deviation, corrective action, and disposition records;calibration records; and microbiological test results. Verification should be performed

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whenever there are indications that a process is unstable or out of control or wheneverthere is a change in product or processing equipment. HACCP plants should berevalidated on a periodic basis, even if no significant changes have occurred in theprocess. In this way, the plan will retain its support base (Bernard, 1997). Examplesof verification activities for fresh-cut lettuce at the water flume wash step are foundin Table 4.8.

ESTABLISH RECORD-KEEPING PROCEDURES (PRINCIPLE 7)

Accurate record keeping is an essential part of any successful HACCP program.Record keeping assures that there is written evidence of all HACCP activities thathave occurred in the plant. HACCP records should be kept in a file separate from qualitycontrol records so that only product safety records are reviewed during HACCP audits.This master file should contain a record of the deliberations (meetings) of theHACCP team and documentation for all aspects of the plan. It should include allHACCP records that have been generated, including justification for the setting ofcritical limits, details on sampling and monitoring procedures, methods of analysis,

FIGURE 4.8 Graphic display of an incapable vs. capable process.

Process Capability Analysis

Min CL Max CL

Outside of limit

Process incapable

Max CLMin CL

Process capable

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corrective actions taken, product dispositions, details on all prerequisite SOPs, exam-ples of all forms, and procedures and reviews of the HACCP plan (Bernard, 1997).The FDA requires that HACCP records be kept on file for at least one year fromthe date of production for refrigerated foods (e.g., fresh-cut produce).

Although record keeping may appear to be a burden, there are some sound reasonsfor this activity, which benefit the processor. First, records provide documentationthat CCPs comply within their CLs to insure product safety. Records are the onlyreference the processor has available for tracing his product once it is distributed inthe marketplace. Records provide a monitoring tool so that process adjustments canbe made before there is loss of process control. Records are the necessary data neededfor regulatory compliance and HACCP auditing by regulator and customers. Recordsprovide irrefutable evidence that proper procedures/processes were followed in strictaccordance with HACCP requirements (Wedding and Stevenson, 1999).

Record keeping includes records that go beyond those that are manufactured ona day-to-day basis. NACMCF (1998) endorses the maintenance of four types of records:

1. Summary of the hazard analysis—including records on the HACCP team’sdeliberations on the rationale for determining hazards and control measures

2. The HACCP plan for each product—including records on the productdescription, distribution and end use, verified flow diagram, and all HACCPplan summaries addressing the seven required components

3. Support documentation—CCP records, CL records monitoring and correc-tive action records, and verification and validation records

4. Daily operational records—including records generated daily and whichreally control the HACCP process for each CCP (Specifically, these includemonitoring, corrective action, and verification.)

Information to be found on various HACCP records can be quite diverse. Forexample, the FDA requires all monitoring records to have the following:

• title of form• name/location of processor• date/time of activity• product identification (including product type, package size, processing

lie, product code where appropriate)• critical limits• actual observations/measurements• operator’s signature or initials• reviewer’s signature or initials• date of the review

An example of a fresh-cut HACCP form for monitoring chlorine/pH levels intomato dump tank water is illustrated in Figure 4.9. Specific records to validate andverify CCP control at the water flume wash step of a fresh-cut lettuce operation arefound in Table 4.8.

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84Fresh

-cut Fru

its and

Vegetab

les: Science, Tech

no

logy, an

d M

arket

Chlorine/pH Daily Monitoring LogS & W Tomato House

Donaldsonville, Georgia

Processing Line: Tomato grading lineSpecific Location: Water in dump tankCritical Limits: Chlorine 150–200 ppm/pH 6.5–7.5

Total Chlorine pH OperatorDate Time 7:15 am 9:30 am 12:15 pm 2:30 pm 7:15 am 12:15 pm Initials

Date Reviewed: ________________________________________________________ Verified: _________________________________________________

FIGURE 4.9 Example of HACCP monitoring record.

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Safety Aspects of Fresh-cut Fruits and Vegetables 85

SUMMARY

To meet the challenges of today’s food safety issues, the FDA has increased itsemphasis toward programs that are proactive and prevention oriented. The mostcomprehensive, science-based program to date for reducing pathogen contaminationin fresh-cut products is HACCP. The HACCP approach focuses on controllingpathogens at their source rather than trying to detect them in finished products. Finalproduct testing is futile, because by this time, pathogens have had the opportunityto cross-contaminate many plant areas and fresh-cut products.

The International Fresh-cut Produce Association (IFPA), which is the tradeassociation for over 530 fresh-cut processors and suppliers in 25 countries, has beenvery active in providing food safety information to its membership. It has publishedfood safety guidelines, designed HACCP models for implementation in fresh-cutoperations, created a yearly technical seminar on current topics, and established atwo- to three-day HACCP workshop for its members. Here, participants learn themechanics of putting plant-specific HACCP programs together. According to a 1997IFPA membership survey, 61% of the respondents had a written, implemented,verifiable HACCP program in place (DeRoever, 1999).

Although useful in preventing and reducing the failure rate at CCPs, the HACCPprogram has an inherent weakness. It gives no advanced warning as to when a CCPhas a high probability of exceeding its CL, thus going out of control. The HACCPprogram’s reliability and effectiveness as a prevention tool can be greatly strength-ened by the incorporation of statistical techniques. SPC can provide an objective,statistically valid means of predicting CCP control during monitoring and verifica-tion activities. Integration of SPC into an HACCP program can only help to validateand verify HACCP performance.

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Physiology of Fresh-cut Fruits and Vegetables

Peter M. A. Toivonen and Jennifer R. DeEll

CONTENTS

IntroductionPhysiological Effects of Cutting on Tissues

Ethylene ProductionRespirationMembrane DeteriorationSecondary Metabolite AccumulationWater LossSusceptibility to Microbiological Spoilage

Factors Affecting Response to CuttingCultivarPreharvest Crop Management Physiological MaturitySeverity or Degree of Cutting-Induced InjuryPre- and Post-Cutting TreatmentsAtmospheric Composition

Consequences of Cutting-Induced Injury on Quality RetentionConclusionReferences

INTRODUCTION

There have been several reviews on the physiology of fresh-cut (minimally pro-cessed) fruits and vegetables (Rolle and Chism, 1987; Watada et al., 1990; Brecht,1995; Watada et al., 1996). This chapter is devoted to the integration of the principlespresented in these reviews and recently published information on fresh-cut fruitsand vegetables.

The fundamental principle underlying quality of fresh-cut fruits and vegetablesis that they are living tissues, and as a consequence, show physiological response

This work was carried out by P. M. A. Toivonen and J. R. DeEll for the Department of Agriculture andAgri-Food, Government of Canada. © Minister of Public Works and Government Services Canada 2001,centre contribution number 2146.

5

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to minimal processing procedures as well to post-processing handling and treatmentsand ultimately to the package environment in which they are enclosed. In addition,the intrinsic physiology and quality of the raw produce will have an influence on theresponse to minimal processing and packaging. Microbial growth is influenced bythe physiology of the minimally processed product, and thus, must also be consid-ered. Physiological processes leading to tissue senescence and deterioration can beminimized or modulated through the implementation of an integrated approachinvolving proper cultivar selection, pre-harvest management, pre- and post-processingtreatments, and application of appropriate packaging that provides optimal atmo-spheres. All of these issues will be discussed in detail, and a summary on theconsequences to quality will be presented.

PHYSIOLOGICAL EFFECTS OF CUTTING ON TISSUES

E

THYLENE

P

RODUCTION

Wounding of plant tissues has long been known to induce ethylene production, andthe time line for the initiation of this response can range anywhere from a fewminutes to an hour after wounding, with maximal rates being produced between6–12 h (Abeles et al., 1992) (Figure 5.1). The potential effects of wound ethyleneare dependent on the type and physiology of the tissue in question. Large increasesin ethylene production, as a consequence of cutting, have been shown in kiwifruit(

Actinidia deliciosa

L.) (Watada et al., 1990; Agar et al., 1999), tomato (

Lycopersiconesculentum

Mill.) (Lee et al., 1970; Mencarelli et al., 1989; Abeles et al., 1992;Brecht, 1995; Artés et al., 1999), winter squash (

Cucurbita maxima

Duch.) (Abeleset al., 1992), papaya (Paull and Chen, 1997), and strawberry (

Fragaria x ananassa

FIGURE 5.1

Effect of wounding on the rate of ethylene production [nl (g FW)

1

h

1

] fromlettuce tissue. All lettuce tissues were incubated at 5

°

C. The vertical error bars represent SEof the mean. (From Ke and Saltveit 1989, used with permission from Munksgaard InternationalPublishers.)

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Physiology of Fresh-cut Fruits and Vegetables

93

Duchesne) (Rosen and Kader, 1989). However, there are some products that responddifferently to wounding. For example, pear (

Pyrus communis

L.) does not show anincrease in ethylene production in response to cutting (Gorny et al., 2000). In anotherreport, cut pear slices were found to have lower ethylene production as comparedwith whole fruit (Rosen and Kader, 1989).

A few contradictory results regarding the effects of wounding on ethylene pro-duction have been reported in bananas (

Musa

spp. AAA) and cantaloupe (

Cucumismelo

L., var. Reticulatus). Slicing of banana has been shown to increase ethyleneproduction in one case (Abe et al., 1998), while no increase in ethylene productionwas observed in another (Watada et al., 1990). The contradictory results may beexplained by the fact that maturity of the bananas was different in the two studies;in the first study, they were at the green tip stage, and in the second study, they wereat a full yellow, post-climacteric stage at the time of cutting. Cutting of cantaloupe inone case (Hoffman and Yang, 1982) resulted in increased ethylene production, butin another study, resulted in a reduction of ethylene production (Luna-Guzmán et al.,1999). Again, the difference can be explained by the fact that wound-induced ethyleneproduction is influenced by fruit maturity; in the first case, the fruits were cut in thepre-climacteric phase, whereas in the second case, the fruits were cut in the post-climacteric phase, when tissue capacity to produce ethylene has declined (Luna-Guzmánet al., 1999). Therefore, maturity of the product (especially for climacteric fruit) mustbe considered in understanding the effect of cutting on ethylene production.

Ethylene production is localized to tissue in the close vicinity of the wound orcutting injury. It has been shown that increase in ethylene production was limitedto within a few millimeters of the cut surface in sliced bananas (Dominguez andVendrell, 1993). The importance of secondary responses of tissues adjacent to thecut cells has been only partially documented (Rolle and Chism, 1987), but this mayexplain the localization of such phenomena as cut surface browning.

Storage temperature also has an effect on wound-induced ethylene production(Figure 5.2). It has been shown that storage of cantaloupe pieces at 0–2.5

°

C willalmost completely suppress wound-induced ethylene as compared to higher storagetemperatures (Madrid and Cantwell, 1993). Similar reductions in ethylene produc-tion for other cut fruits and vegetables would be expected at low post-cutting storagetemperatures.

R

ESPIRATION

Wounding results in increases in respiration, but the initiation of this response isdelayed compared to that found for wound-induced ethylene (Brecht, 1995). Increasesin respiration in response to cutting may depend on the commodity under consider-ation, since bananas do not appear to show an increase but kiwifruit do (Figure 5.3).The increase in respiration has been assumed to be due to enhanced aerobic mitochon-drial respiration. This assumption is supported by the fact that changes in mitochondrialstructure and increases in their numbers and function have been shown to be inducedby wounding (Asahi, 1978).

Respiration rate is associated with product shelf life potential, with high ratesof respiration being correlated with short shelf life (Kader, 1987). Therefore, it has

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FIGURE 5.2

C

2

H

4

production of whole and fresh-cut ‘Durinta’ tomato at 2 and 10

°

C duringthe first hours after slicing and up to seven days. Each point represents the mean of six wholefruits or six fresh-cut fruits. Vertical lines represent S.D.s. (Reprinted from Artés et al. 1999,

Postharvest Biology and Technology,

Vol. 17, pp. 153–162, with permission from ElsevierScience.)

FIGURE 5.3

Respiration rates of sliced and whole banana and kiwifruit held at 20

°

C. Bananais sliced to 4-cm length sections and kiwifruit to 1-cm thick slices. (From Watada et al. 1990,used with permission from the Institute of Food Technologists.)

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been assumed that increases in respiration brought about by cutting are expected toresult in shorter shelf life (Rolle and Chism, 1987). A wide range of fresh-cutproducts shows significant increases in respiration, and generally, this effect is onlyseen when the cut product is stored at higher temperatures (Watada et al., 1996).There are some exceptions—pear and strawberry slices have been shown to respireat a greater rate than whole fruits at both 2.5 and 20

°

C (Rosen and Kader, 1989).Gorny et al. (2000) did not find an immediate effect of cutting on respiration ofpear, but respiration rates in slices rose after six days at 10

°

C. The delayed increasein respiration in response to cutting may have been related to the growth of microor-ganisms on the cut slices, as has been suggested in other fresh-cut products (Varoquauxet al., 1996a).

Increases in respiration in response to cutting may be quite substantial in somecases. Slicing of mature green tomatoes results in increased respiration by up to40% when stored at 8

°

C, as compared to intact product (Mencarelli et al., 1989).Many other fruits and vegetables show such large increases in respiration, mainlywhen stored at higher temperatures (Watada et al., 1996) (see Figures 5.4 and 5.5).

The basis for the rise in respiration may not always be completely explainedby an enhancement in aerobic respiration. It has been demonstrated in cut potatoesthat the rise in respiration after cutting or wounding is at least partially a resultof

α

-oxidation of long-chain fatty acids (Martin and Stumpf, 1959; Laties, 1964;

FIGURE 5.4

Respiration rates of intact and shredded cabbage stored at 2.5

°

C (36.5

°

F), 5

°

C(41

°

F), 7.5

°

C (45

°

F), and 10

°

C (50

°

F). The intact heads of cabbage had been harvested,cooled, and processed the same day. (From Cantwell 1992, used with permission from theUniversity of California Board of Regents.)

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Laties et al., 1972). Increases in O

2

consumption associated with

α

-oxidation coin-cide with membrane deteriorative processes, which will be described in a separatediscussion on membrane deterioration.

Increases in respiration in response to cutting might be expected to be at leastpartially explained by removal of barriers (i.e., periderm or cuticle) to gas exchangein the tissues. However, it has been shown in apples that minimal processing doesnot contribute to increased respiration due to reduction of resistance to diffusion forO

2

(Lakakul et al., 1999). Therefore, the physical removal of gas diffusion barriersmay not be an important factor in the physiological response to cutting.

Another aspect of respiration and minimal processing is that of susceptibility toanaerobic metabolism. When cut product is placed into modified atmosphere pack-aging, it is exposed to high CO

2

and/or low O

2

, and the sensitivity of cut productto modified atmospheres may be quite different than for whole product. Cut lettucetissues are less susceptible to developing fermentative metabolism than whole headswhen exposed to high CO

2

(Mateos et al., 1993). In contrast, shredded carrots aremore susceptible to developing anaerobic metabolism than whole carrots, and thismay be associated with the tissues’ requirement to supply the ATP requirements toensure cell survival (Rolle and Chism, 1987).

FIGURE 5.5

Respiration rate of whole and fresh-cut ‘Durinta’ tomato at 2 and 10

°

C duringthe first hours after slicing and up to seven days. Each point represents the mean of six wholefruits or six fresh-cut fruits. Vertical lines represent S.D.s. (Reprinted from Artés et al. 1999,

Postharvest Biology and Technology,

Vol. 17, pp. 153–162, with permission from ElsevierScience.)

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The level of anaerobic metabolism is determined by the O

2

threshold for anaerobicmetabolism induction, as well as the handling temperature of the fruit or vegetableproduct (Lakakul et al., 1999). The target atmosphere for real-life distribution mustreflect a potential for exposure to non-ideal temperatures, because package O

2

atmo-spheres are generally designed to be low at ideal temperatures (e.g., 0

°

C). If thepackages are exposed to higher temperatures during distribution, there is a risk ofanaerobic metabolite accumulation. The risk of off-flavors in response to anaerobicmetabolite accumulation is high in some fruit cultivars, whereas in others, the riskis much lower (Ke et al., 1991). In some commodities, such as iceberg lettuce, wherea flavored dressing is used by the consumer of the product, some accumulations ofanaerobic metabolites might be tolerated (Smyth et al., 1998).

M

EMBRANE

D

ETERIORATION

Membrane deterioration results in decompartmentation of cellular structure and orga-nization and loss of normal cellular function. Many secondary events are a consequenceof membrane deterioration, the most commonly recognized being tissue browning(Rolle and Chism, 1987; Brecht, 1995). Another is the development of off-odors (Brecht,1995). Wounding of tissues can result in relatively rapid deterioration in membranes,and this has been associated with oxygen-free radical production in response towounding (Thompson et al., 1987). In potato, wounding has been shown to rapidlycause membrane lipid breakdown (Galliard, 1970). Wounding has also been shownto result in enzymatic degradation of membrane components. Induction of lipid acylhydrolase (Wardale and Galliard, 1977) and phospholipase D (Galliard, 1979) activ-ities result in the production of free fatty acid from membrane lipids. These liberatedfatty acids can disrupt the cellular function via direct lysis of organelles and throughbinding to and subsequent inactivation of proteins (Galliard, 1979). The free fattyacids are also subject to oxidation via either

α

-oxidation (Galliard and Matthews, 1976;Laties et al., 1972) or lipoxygenase activity (Galliard and Phillips, 1976). Woundrespiration has been at least partially attributed to the

α

-oxidation of fatty acids inpotato tissues (Laties and Hoelle, 1967). Ethylene production as a consequence ofmetabolism of free fatty acids by lipoxygenase has been demonstrated in tomato(Sheng et al., 2000), suggesting that the wound-induced membrane breakdown maybe associated with wound-induced ethylene production. However, not all fruits andvegetables show a wound-induced membrane lipid breakdown, with carrot, avocado,and banana being notable examples (Theologis and Laties, 1980).

S

ECONDARY

M

ETABOLITE

A

CCUMULATION

Phenolic accumulation is one of the most studied phenomena in response to wounding.Wounding has two effects on phenolic metabolism (Rhodes and Wooltorton, 1978).The first is the oxidation of endogenous phenolics as a consequence of cell membranebreakdown, allowing the mixing of the phenolics with oxidative enzyme systems, whichare normally separated by membranes. The second is the stimulation of cells adjacentto the injury to produce more phenolics in an attempt to initiate repair processes (i.e.,lignification). Phenolic accumulation is initiated via increased activities in phenylalanine

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ammonia lyase activity, and wounding has been clearly shown to increase the activityof this enzyme in iceberg lettuce (Figure 5.6). Chlorogenic acid can also accumulatein shredded carrots when stored in air (Babic et al., 1993). Isocoumarin levels increasein response to ethylene exposure, cutting, or bruising in carrots (Lafuente et al., 1996).This response is not seen in cut-and-peel (‘baby’) carrots though, likely due to the factthat most of the phenolic metabolism is localized in the peel tissue (Sarkar and Phan,1979), which is removed in the cut-and-peel process. However, if carrots are bruisedor exposed to ethylene prior to processing, then isocoumarin accumulations will occur,affecting quality of cut-and-peel product. Wounding increased phenolic acids andanthocyanins in midrib tissues of red pigmented lettuce; however, there was no signif-icant effect in the green and red tissues (Ferreres et al., 1997). Onion tissue, whendisrupted, will enzymatically produce a bitter compound (either a triterpenoid orflavonoid) over time, and its accumulation can be controlled by acidification(Schwimmer, 1967). Precursors to a pink pigment accumulate in response to wound-induced alliinase activity (Shannon et al., 1967). These precursors react with free aminoacids and carbonyls to develop a pink pigment in cut onion tissues.

Sulfur-containing compounds can also accumulate in response to wounding. Severalunpleasant sulfur compounds can increase with time after cutting in cabbage tissues,such as methanethiol and dimethyl disulfide (Chin and Lindsay, 1993). The accu-mulation of such compounds is known to be associated with membrane deterioration.The loss of cellular compartmentation allows enzymes such as cysteine sulfoxidelyase to come into contact with various sulfur-containing substrates and oxidizethem into these unpleasant sulfur volatiles (Dan et al., 1997). Allyl isothiocyanatescan also accumulate in response to cutting in shredded cabbage (Yano et al., 1986).

Tissue disruption of green bell peppers results in the rapid production of six-carbon aldehydes and alcohols, which are a consequence of the oxidation of free

FIGURE 5.6

Changes in wound-induced PAL activity [

µ

mol (g FW)

1

h

1

] as affected bydistance from the wound. The numbers in the parentheses represent the distance from thewounded surface. The vertical error bars represent SE of the mean. (From Ke and Saltveit1989, used with permission from Munksgaard International Publishers.)

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99

fatty acids that have been enzymatically (by lipoxygenase and hydroperoxide lyase)released from membranes (Wu and Liou, 1986). Some of these compounds areassociated with off-flavors in peppers.

Accumulation of long-chain aliphatic compounds (fatty acids or alcohols) thatcomprise suberin polymers occurs in response to wounding in tomato fruit (

Lycopersiconesculentum

Mill.) and bean pods (

Phaseolus vulgaris

L.) (Dean and Kolattukudy,1976). The production of suberin is an important part of the process leading to woundhealing.

W

ATER

L

OSS

Water loss in fruits and vegetables is determined by many factors, probably the mostimportant being the resistance of outer periderm or cuticle to transpirational move-ment of water vapor (Ben-Yehoshua, 1987). However, peeling and cutting result inreduction or elimination of the resistance by these barriers to transpiration. Twoissues are important to water loss: reduction of tissue bulk, i.e., increase in surfacearea to volume ratio, and the removal of protective periderm tissues. Both mecha-nisms for resultant increases in water loss, are demonstrated by the fact that slicingof kiwifruit results in increased rates of water loss, and subsequent peel removalfrom the slices results in a further increase in weight loss (Figure 5.7). Increasedrates of water loss result in greater susceptibility to wilting and/or shriveling. Slicingof pears resulted in high rates of moisture loss from cut surfaces, which was animportant factor in quality loss (Gorny et al., 2000). Peeled ‘Majestic’ potatoes havea water loss of 3.3–3.9 mg H

2

O cm

2

mbar wpd

1

h

1

, while nonpeeled, cured potatoeshave a moisture loss rate of 0.007 mg H

2

O cm

2

mbar wpd

1

h

1

(Ben-Yehoshua, 1987).The peeling method of carrots influences the water loss of subsequently processed

fresh slices. Coarse abrasion peeling results in three times greater weight loss of

FIGURE 5.7

Effect of wounding on mass loss of whole kiwifruit, whole-peeled kiwifruit,peeled slices, and unpeeled slices stored at 20

°

C for three days. (From Agar et al. 1999, usedwith permission from the Institute of Food Technologists.)

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packaged slices as compared to slices made from hand-peeled carrots (Barry-Ryanand O’Beirne, 2000). Slices made from carrots that had been abrasion-peeled usinga fine peeling plate had intermediate levels of water loss. The slicing process can alsoinfluence the water loss in carrot slices, with machine-sliced product losing water 30%faster than manually razor-sliced product (Barry-Ryan and O’Beirne, 1998).

S

USCEPTIBILITY

TO

M

ICROBIOLOGICAL

S

POILAGE

A wide variety of microorganisms have been found to be actively growing on pack-aged, minimally processed fruits and vegetables, and these include mesophilic bacteria,lactic acid bacteria, coliforms (some fecal in origin), and yeasts and molds (Nguyen-Theand Carlin, 1994). Increases in microbial populations on minimally processed productsare often associated with apparent increases in respiration rates with time in storage(Figure 5.8). Tissue decay is closely associated with microbial activity (aerobic andlactic bacteria). Spoilage of packaged bean sprouts was associated with the reductionof O

2

levels in the package, and the accumulation of acetate and lactate linked thespoilage with microbial activity (Varoquaux et al., 1996a). Many microorganisms

FIGURE 5.8

Changes in (a) oxygen uptake rates and (b) total aerobic counts during storageof bean sprouts at various temperatures. Results are expressed as mmol h

1

kg

1

and cfu g

1

of fresh weight, respectively. (From Varoquaux et al. 1996a, Copyright Society of ChemicalIndustry. Reproduced with permission. Permission is granted by John Wiley & Sons Ltd. onbehalf of the SCI.)

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produce pectin degrading enzymes, which lead to tissue softening and breakdown.However, relatively high microbial counts can be found on what would be consideredacceptable quality product (Watada et al., 1996). In some products, microbiologicalspoilage does not appear to be of great concern. For example, it has been reportedthat packaged minimally processed Chinese cabbage does not show any microbialspoilage even after 21 days of storage at 5

°

C (Kim and Klieber, 1997).The largest populations of microorganisms are found within broken cells or in

tissues adjacent to broken cells after packaged fresh-cut product has been stored(Watada et al., 1996). Presumably, the damaged tissue and broken cells providenutrients and a protected environment for growth of most types of microflora. Thisis demonstrated by the fact that microbial growth is much greater on minimallyprocessed product as compared with intact product. For example, cut lettuce hasgenerally higher microbial count than intact heads (Priepke et al., 1976). In addition,higher populations of microbes have been associated with faster rates of tissue decayin minimally processed product (King and Bolin, 1989; King et al., 1991).

The tissue from which the minimally processed product is derived can determinethe rate of growth of microbial population. Inner (younger) leaves of endive have smallerpopulations of microbes, which might be explained by the effect of protective outerleaves (Jacques and Morris, 1995). However, the growth of bacteria inoculated onthese inner leaves is inhibited as compared with outer (older) leaves. This suggeststhat there is a physiological basis for the resistance of such tissue to bacterial growth.

FACTORS AFFECTING RESPONSE TO CUTTING

C

ULTIVAR

Romig (1995) discussed the importance of the appropriate selection and developmentof fruit and vegetable cultivars specifically for use in fresh-cut products. The effectof cultivar selection on the raw product initial physiology and quality was consideredto have a subsequent impact on the acceptability of a packaged, fresh-cut fruit orvegetable product in the retail market. In a few cases, traits that could be improvedthrough conventional breeding and/or genetic transformation have been selected forcertain vegetables (Romig, 1995). However, the literature does not show any specificsuccesses in this area. There have been numerous reports regarding identification ofcultivar differences, and these will be dealt with in the following discussion.

Different cabbage cultivars will produce different levels of various sulfur volatilesin response to injury (Chin and Lindsay, 1993). Some cultivars produce more dimethyldisulfide and dimethyl trisulfide in response to cutting, and these compounds areconsidered unpleasant. This information would, therefore, suggest that cultivars thatproduce large amounts of these volatiles on cutting should not be used for freshcabbage salads. Different cabbage cultivars also produce different levels of allylisothiocyanates, and their accumulation accounts for reductions in rates of browningand ethylene production of the shredded product (Yano et al., 1986; Nagata, 1996).

There are no differences in wound-induced phenolic accumulation and browningof ‘Baby’ and ‘Romaine’ lettuces (Castañer et al., 1999). In butterhead lettuce, thecultivar ‘Ritmo’ produced higher CO

2

concentrations in 30

µ

m thick polypropylene

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packages than ‘Musette’ or ‘Nancy’, and it was also more susceptible to tissue injuryin the package in response to high CO

2

, resulting in greater levels of tissue browning(Varoquaux et al., 1996b). Minimally processed ‘Calmar’ and ‘Sea Green’ iceberglettuces were the least susceptible to browning out of eight tested, and ‘Nerone’ wasthe most susceptible after six days of storage at 5

°

C (Couture et al., 1993). Thereare also differences in wounding-induced accumulations of phenolics that are depen-dent on cultivar for shredded carrot (Babic et al., 1993).

No single apple cultivar tested in New York state showed an overall bettersuitability for use as packaged fresh slices. The numbered cultivar ‘NY 674’ devel-oped the least browning of all 12 tested, however, it was the least firm of all thecultivars (Kim et al., 1993b). In contrast, ‘Liberty’ was the firmest cultivar, but itwas susceptible to cut-surface browning. It was concluded that ‘NY 674’, ‘Cortland’,‘Golden Delicious’, ‘Empire’, and ‘Delicious’ were considered to be acceptable forfresh slices, whereas ‘Mutsu’ and ‘Rome’ were poorly suited for this purpose. Therewere differences in respiration in slices made from the 12 cultivars, but these werenot related to suitability for packaged, fresh slices (Kim et al., 1993b).

There is limited evidence that there are cultivar differences in rates of membranedeterioration in response to minimal processing. Membrane deterioration in shreddedcarrots in response to the shredding process is dependent on cultivar, with ‘Caropak’showing greater rates of membrane deterioration than ‘Apache’ (Picchioni and Watada,1998).

The accumulation of fermentation products in response to high CO

2

can beinfluenced by the cultivar of the fruit or vegetable under consideration. High CO

2

atmospheres in package atmospheres have been suggested as a good approach forpreserving fresh-cut strawberry slice quality (Rosen and Kader, 1989). However, ithas been found that there is a great variability in response of strawberry cultivarsto high CO

2

(Watkins et al., 1999). In addition, Watkins et al. (1999) found that thedifferent cultivars showed different degrees of firmness improvement under highCO

2

atmospheres. Similar differences in response of packaged raspberry cultivarsto package CO

2

levels have been demonstrated (Toivonen et al., 2000).There are large differences in shelf life potential for peach and nectarine slices

made from different cultivars (Gorny et al., 1999). It is assumed that these differencesrelate to differences in ripening-related physiology of the cultivars.

Susceptibility to microbial spoilage can also be influenced by cultivar. The shelflife of different cultivars of packaged spinach was found to be dependent on therainfall conditions during production (Johnson et al., 1989). The cultivars ‘Seven R’and ‘Grandstand’ had the best shelf life under normal rainfall conditions, whereas‘Gladiator’ and ‘Melody’ had the best shelf life under high rainfall conditions. Thedifferences in shelf life were primarily associated with differences in susceptibilityto microbial decay in the packaged product.

P

REHARVEST

C

ROP

M

ANAGEMENT

Very little information has been published in respect to preharvest crop managementon the postharvest physiology of fresh-cut fruits and vegetables. A series of reviewson preharvest factors and their effects on fruits and vegetables have been published

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recently (Crisosto et al., 1997; Prange and DeEll, 1997; Weston and Barth, 1997),and some of the discussions in these reviews are pertinent here. Good pest anddisease management may be the most important preharvest factor affecting thequality of fresh-cut fruit and vegetable products. Other issues that appear to beconsistently important to fruit and vegetable quality are irrigation and calciumnutrition. Rates of irrigation and calcium nutrition have effects on postharvest decayand on tissue firmness. Excess irrigation results in development of tissues that aresusceptible to bruising and injury (Prange and DeEll, 1997). In some cases, thereare physiological responses to nitrogen and phosphorus fertilization (Prange andDeEll, 1997; Weston and Barth, 1997), however, these are often overshadowed bythe effects of climatic variations (Prange and DeEll, 1997). Excess irrigation willalso reduce the soluble solids content of fruit and vegetable tissues (Crisosto et al.,1997; Prange and DeEll, 1997; Weston and Barth, 1997), and this may have an effecton respiration rates (Blanchard et al., 1996).

The climatic conditions in which fruits and vegetables are produced can have asignificant influence on fresh-cut product quality. Muskmelon that is shaded beforeharvest can have lower sucrose levels and higher acetaldehyde and ethanol levels,and this can lead to “water-soaked” flesh tissues. This particular problem has beenshown to occur under dull, cool summer growing conditions (Nishizawa et al., 1998).Carrots of the same cultivar grown in different geographical regions will producedifferent levels of phenolics in response to shredding (Babic et al., 1993); however,the basis of these differences are not understood. Growing region also has an effecton shelf life of pear slices (Gorny et al., 2000) as related to effects on browning andsoftening, but again, these effects are not understood.

There is very little information on crop nutrition on the physiological responseto cutting in minimally processed fruits and vegetables. Calcium is the best studiedof all the crop nutrients in terms of postharvest quality (Fallahi et al., 1997). Ingeneral, preharvest calcium nutrition improves firmness retention and delays mem-brane deterioration and ripening in whole fruits. Recent work is showing that pre-harvest calcium applications can improve the firmness retention in green and coloredpeppers (Toivonen, unpublished data). Further work is required in this area, becauseimprovements in nutrition may have tremendous impacts on the physiological responseto minimal processing.

PHYSIOLOGICAL MATURITY

The physiological maturity of fruits or vegetables is known to impact the woundingresponse. This is especially true for climacteric fruits. Cantaloupes harvested early( slip) had better quality retention (decay/discoloration) than those harvested atlate maturity (full slip) (Madrid and Cantwell, 1993). Pieces cut from the slipfruit produced less ethylene, had lower respiration rates, and stayed firmer than thosecut from full slip fruits. Slices from partially colored or fully colored bell peppersretained better quality over 12 days in controlled atmospheres than slices from greenbell peppers (López-Gálvez et al., 1997a). This is opposite to results found withwhole peppers where fully ripe (red) fruit deteriorated much more quickly thanimmature (green) fruit (Lurie and Ben-Yehoshua, 1986). Immature carrots produce

14---

14---

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104 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

greater amounts of isocoumarin, a compound responsible for bitterness, in responseto ethylene than mature carrots (Lafuente et al., 1996). Minimally processed imma-ture iceberg lettuce was less susceptible to browning than mature or overmaturelettuce (Couture et al., 1993). Therefore, while most fruits and vegetables are bettersuited to minimal processing in less mature physiological stages, some productssuch as bell peppers may be most suitable at more advanced stages of maturity.

Papaya fruit at 55–80% skin yellowing were found to be the most suitable forminimal processing (Paull and Chen, 1997). Fruit with less than 55% skin yellowingshowed greater increases in ethylene production and respiration in response to slicingand de-seeding, and the flesh of processed product was not soft enough to beacceptable for consumption. At the other end of the spectrum, papaya fruit whichwere full yellow at cutting showed little increase in ethylene or respiration, and thepieces were easily bruised and too soft to handle.

Pears with 44–58 N firmness were found to be optimal for quality of fresh slices(Gorny et al., 2000), and small fruit (122–135 g) were more susceptible to browningthan large fruits (152 g). Peaches and nectarines at a firmness of 18–31 N werefound to be optimal for packaged fresh slices (Gorny et al., 1999), however, notmuch information was given on the maturity characteristics at picking.

In areas where root crops can be left in the soil and harvested throughout thewinter (e.g., Ireland), carrots that are harvested after the winter produce slices thatbreak down faster than carrots that are harvested earlier in the fall (Barry-Ryan andO’Beirne, 2000). This is presumably due to the higher microbial loads on the carrotsharvested after the winter. Similar findings were reported for shredded carrots byBabic et al. (1992). Therefore, root crops used for fresh-cut products should beharvested as soon as they mature.

SEVERITY OR DEGREE OF CUTTING-INDUCED INJURY

The severity of cutting can have a large influence on ethylene production andrespiration rates (Figure 5.9). In bananas, it was found that as the angle of cut wasincreased, total cut area increased, and this resulted in parallel increases in respirationrates and ethylene production (Abe et al., 1998). These differences in respirationand ethylene production were related to shelf life of the sliced bananas. In anotherstudy, cut potatoes were found to have twice the respiration rate of whole-peeledpotatoes that, in turn, have double the respiration of intact unpeeled potatoes (Gunesand Lee, 1997).

Susceptibility to anaerobic metabolism can also be affected by severe cuttingprocedures. For example, carrot shreds show a significant accumulation of ethanoland acetaldehyde even in 2% O2, which is not considered to be at the anaerobicthreshold (Kato-Noguchi and Watada, 1997a), suggesting the extensive injury to thetissues induces anaerobic metabolism in order to supply the ATP requirements forcell survival (Rolle and Chism, 1987).

Phenolic metabolism is also affected by the severity of the processing method.As tissue injury levels increase, so does phenylalanine ammonia lyase (PAL) activityin the tissue (Figure 5.10). This results in increased accumulations of phenolics inproducts such as lettuce (Ke and Saltveit, 1989). Severity of abrasion on the surfaceof peeled carrots affects the degree of lignification of the surface, and use of a sharp

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Physiology of Fresh-cut Fruits and Vegetables 105

FIGURE 5.9 Effect of wounding on the C2H4 (a) and CO2 (b) production rates of wholekiwifruit, whole-peeled fruit, peel, peeled, and unpeeled fruit slices stored at 20°C for 6 h.(From Agar et al. 1999, used with permission from the Institute of Food Technologists.)

FIGURE 5.10 Phenylalanine ammonia lyase (PAL) activity [µmol (g FW−1 h−1)] as relatedto the degree of wounding. Wounding was done by uniformly puncturing an 8 cm2 area ofmidrib tissue with a sterile 26-gauge hypodermic needle. All measurements were taken onthe second day of incubation. The vertical error bars represent SE (standard error) of themean. (From Ke and Saltveit 1989, used with permission from Munksgaard InternationalPublishers.)

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106 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

blade completely prevents the lignification even at 35 days of post-process storageat 2°C (Bolin and Huxsoll, 1991). However, if much of the outer carrot tissue isremoved with the peeling process, there is less problem with lignification, which islikely due to the fact that metabolic machinery to produce lignification is localizedin the outer peel tissue (Sarkar and Phan, 1979).

The type of peeling or cutting process can also influence the degree of physio-logical response by tissues. Fine abrasion peeling results in lower weight loss ofpackaged slices made from the peeled carrots as compared with coarse abrasionpeeling, which causes more tissue injury (Figure 5.11). In addition, the respirationrates of slices made from fine abrasion-peeled carrots were significantly lower than forslices made from coarse abrasion-peeled carrots. Carrots that were hand-peeled witha sharp blade (which causes the least amount of tissue damage) exhibited lowerwater loss, respiration, and microbial counts than slices made from either fine orcoarse abrasion-peeled carrots. The severity of injury from the slicing procedurealso has effects on water loss and microbial growth in sliced carrots (Barry-Ryanand O’Beirne, 1998). Machine slicing as opposed to manual slicing with a sharprazor blade results in more bacterial, yeast, and mold growth in packaged slicedcarrots. Weight loss also increases by ∼30% in the machine-sliced product as com-pared with the manually sliced product. This difference relates to the degree of tissueinjury induced by the machine slicer (Barry-Ryan and O’Beirne, 1998). Hand-peeling with a sharp blade and lye peeling resulted in less surface browning thanabrasion peeling in potatoes (Gunes and Lee, 1997). Enzymatic peeling to produceminimally processed orange (Citrus sinensis) segments resulted in half the weightloss than for segments made from manually peeled oranges, presumably due to thereduction of injury to the segments with enzymatic peeling (Pretel et al., 1998).

FIGURE 5.11 Effects of peeling method on weight loss of carrot disks during storage at8°C. Values are means for four determinations, each done in duplicate, separated by Fisher’sleast significant difference (P < 0.05), denoted by different letters. (From Barry-Ryan andO’Beirne 2000, used with permission from Blackwell Science Ltd.)

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Physiology of Fresh-cut Fruits and Vegetables 107

Manual peeling resulted in greater bacterial growth than enzymatic peeling for theseorange segments.

PRE- AND POST-CUTTING TREATMENTS

One of the most studied post-cutting treatments is the use of calcium dips. CaCl2

(1% w/v) has been shown to prevent softening of strawberry and pear slices, espe-cially when combined with modified atmospheres (Rosen and Kader, 1989). CaCl2

was also effective in slowing ripening and softening in sliced tomato when packagedslices were stored at 2°C, but not at 10°C (Artés et al., 1999). CaCl2 at concentrationsbetween 1 and 5% (w/v) suppressed wound-induced respiration in fresh-cut canta-loupe but did not have any effect on ethylene production (Luna-Guzmán et al., 1999;Luna-Guzmán and Barrett, 2000). CaCl2 dips at a 0.05 M concentration delayed thecatabolism of membrane phospholipids in cabbage leaf tissue (Chéour et al., 1992),delaying senescence in the tissues. CaCl2 dips enhance the maintenance of membranestructure and function in shredded carrots (Picchioni and Watada, 1998). Calciumlactate dips can increase the shelf life of peach slices via effects on firmness retention(Gorny et al., 1999).

Organic acids such as citric acid have also been used to control physiologicalchanges in fresh-cut tissues. Citric acid dips (i.e., low pH) will reduce peeling-induced surface lignification in minimally processed cut-and-peel carrots, likely dueto the inactivation of the enzymes that are responsible for processes leading tolignification (Bolin and Huxsoll, 1991). Citric acid dips of 1 mM or higher concen-tration reduce the respiratory rate of shredded carrots by 50% or more (Kato-Noguchiand Watada, 1997b).

Ascorbic acid (vitamin C) is a reducing agent often used to prevent oxidationreactions such as browning; however, there may be effects on other physiologicalprocesses in the cut tissues. Ascorbic acid dips reduced the respiration of ‘Fuji’apple slices stored in a 0% O2 atmosphere (Gil et al., 1998). In air atmosphere, theascorbic acid dips reduced ethylene production and increased the respiration ofapples slices (Gil et al., 1998). Browning of slices was reduced in both air and 0%O2 atmospheres.

The use of mild heat treatments has been found to have profound physiologicaleffects on fresh-cut fruit and vegetable products. Heat shock treatments of 45°C for120 s, 50°C for 60 s, or 55°C for 30 s reduced PAL activity that resulted in lesswound-induced phenolic accumulation in iceberg lettuce (Loaiza-Velarde et al.,1997). Pre-cutting heat treatments (45°C) have been shown to reduce browning andenhance firmness retention of slices made from treated apples (Kim et al., 1993a).However, not all cultivars respond favorably to heat treatments—only ‘Delicious’and ‘Golden Delicious’ showed a significant benefit for slices after eight days ofstorage at 2°C. In contrast, other cultivars (e.g., ‘Liberty’, ‘Munroe’, ‘Rome’, and‘RI Greening’) showed increased browning in response to the heat treatments (Kimet al., 1993a). The mechanism of the response to heat treatments relates to theireffect on physiological processes; heat treatment inhibits ethylene synthesis, tissueresponse to ethylene, and cell wall degradation associated with hydrolytic enzymessuch as polygalacturonase and galactosidases (Lurie and Klein, 1990; Lurie, 1998).

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While respiration is initially enhanced, it subsequently falls below the level foundin nontreated whole products. These effects may explain why heat treatments inhibitcutting-induced changes in fruits and vegetables. Heat treatments up to 60°C for 1min also improved CaCl2 uptake and firmness retention in fresh-cut cantaloupe(Luna-Guzmán et al., 1999).

The postharvest storage method and/or duration can be considered postharvesttreatments and have been, in a few cases, found to influence post-cutting physiology.Storage for 30 days will reduce isocoumarin accumulation in response to ethyleneby severalfold in fresh-cut carrots, as compared to freshly harvested carrots (Lafuenteet al., 1996). Freshly harvested pears and those stored in CA (2% O2, 98% N2)produced better quality slices than those stored in air (Gorny et al., 2000).

Edible coatings offer several possible benefits to fresh-cut fruits and vegetables.Such coatings can provide a modified atmosphere for the cut pieces, and thus, canreduce water loss from cut surfaces. With the incorporation of additives or preserva-tives, they can control cut-surface browning and microbial growth on the damagedtissues (Baldwin et al., 1995). This technology is still emerging, but there have beenseveral successful developments using an array of materials, including lipids, polysac-charides, and/or proteins as the base components in the coatings. Functional additivesare expected to improve the benefits from using these coatings. Potential preservativeadditives that are being considered are benzoic acid, sodium benzoate, sorbic acid,potassium sorbate, and propionic acid (Baldwin et al., 1995). Potential antioxidantadditives include ascorbic acid, citric acid, phosphoric acid, and other compounds.

A cellulose-based edible coating applied to cut apples and potatoes was mosteffective in controlling moisture loss when the formulation contained soy protein(Baldwin et al., 1996). The addition of ascorbic acid to the formulation delayedsurface browning, while the addition of sodium benzoate or potassium sorbate helpedto control microbial growth. Adjustment of pH to 2.5 resulted in optimal control ofboth browning and microbial growth (Baldwin et al., 1996). A wrap made fromedible film composed of fruit puree and lipid material was found to be an effectiveapproach to controlling moisture loss and surface browning in apple slices (McHughand Senesi, 2000).

Low-dose ionizing irradiation is being investigated as an approach to sanitizefresh-cut fruit and vegetable products. The effects of such treatments on physiologyand quality of fruit and vegetable tissue must be ascertained to determine theacceptability of this approach to product sanitation. Low-dose irradiation (0.19 kGy)significantly reduces microbial populations in packaged, cut iceberg lettuce andmoderately increases respiration (Hagenmaier and Baker, 1997). Overall, the qualityof the packaged cut lettuce was improved by the irradiation treatment. The use of2 kGy of irradiation increased the respiration of shredded carrots but slowed theloss of sugars and inhibited the growth of aerobic mesophilic and lactic acid bacteria(Chervin and Boisseau, 1994). The quality of irradiated shredded carrots was muchhigher than those sanitized using conventional industry practice (i.e., chlorinewashes). Hagenmaier and Baker (1998) found that a 0.5 kGy dose of gamma irradiationhad only a minor effect on respiration but significantly reduced the microbial populationin shredded carrots as compared with chlorine-washed shredded carrots. This effectwas persistent over nine days of storage in modified atmosphere packaging (MAP).

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Physiology of Fresh-cut Fruits and Vegetables 109

Doses of up to 2.4 kGy reduced ethylene production and caused a transitory increasein respiration of apple slices (Gunes et al., 2000). Softening of the tissues resultedfrom doses above 0.5 kGy, however, it is not known whether the level of softeningis commercially important.

ATMOSPHERIC COMPOSITION

The basic principle for the use of packaged, fresh-cut products is that modifiedatmospheres are theoretically expected to control physiological and quality changesin the product. Low O2 and high CO2 are known to inhibit ethylene action (Burgand Burg, 1967) (Figure 5.12). For some fruits or vegetables, O2 is the main factorthat controls respiration. As an example, respiration rates in packaged carrot cubeswere dependent on the O2 levels in the package but were not affected by the CO2

levels (Sode and Kühn, 1998). The level of respiration suppression that a controlledatmosphere may cause is dependent on the product. Furthermore, the sensitivity ofrespiration to elevated CO2 and reduced O2 levels depends on the commodity in ques-tion. The respiration of cut carrots appears to be unaffected by CO2 levels, as theyincrease well above 21%. On the other hand, cut lettuce in a relatively impermeablecontainer appears to maintain a balance of 21% between CO2 and O2 (Priepke et al.,1976). Lettuce respiration slows as the package atmosphere becomes more modified,suggesting that cut lettuce is less susceptible to the development of anaerobic metab-olism than is cut carrot. Celery behaves similarly to carrot, whereas radish, greenonion, and endive behave similarly to lettuce. Similar differences are seen in fresh-cut fruits. It was shown that an atmosphere of 2% O2 and 10% CO2 significantly

FIGURE 5.12 Effect of external oxygen levels on the response of pea seedlings to ethylene.Inset: the effect of internal O2 on the concentration of ethylene required to give a responsehalf that of the maximal response KA. (Reprinted from Beaudry 1999, Postharvest Biologyand Technology, Vol. 15, pp. 293–303, with permission from Elsevier Science.)

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reduced respiration and ethylene production in cut honeydew melons but had a muchsmaller effect on cut strawberries and peaches (Qi and Watada, 1997). Therefore, itmust be recognized that controlled or modified atmospheres may not have consistentor predictable effects on CO2 production and O2 uptake in different types of tissues,and this lack of consistent response is further complicated by the effect of storagetemperature (Table 5.1).

Due to the lack of predictability of response to atmospheres for the varying rangeof products, the potential effects of modified atmospheres on a specific fruit orvegetable product must, therefore, be considered on an individual basis. For example,an atmosphere containing 0.5% O2 was found to be effective in preventing browningand softening in sliced pears, whereas an atmosphere containing 12% CO2 preventedsoftening in strawberries (Rosen and Kader, 1989). In contrast, high CO2 and lowO2 atmospheres both have detrimental effects on quality of shredded carrots—highCO2 leads to enhancement of lactic bacteria growth, and low O2 leads to qualitydeterioration (flavor and texture) (Barry-Ryan et al., 2000).

TABLE 5.1 Carbon Dioxide Production and Oxygen Uptake Rates of Fresh-cut Products Stored in Air and Controlled Atmospheres

Commodity °°°°C AtmosphereCO2 Production

(ml kg−1 h−1)O2 Uptake

(ml kg−1 h−1)Kiwifruit slices 0 Air 2.1 2.8

1% O2 + 5% CO2 2.3 1.85 Air 2.4 2.6

1% O2 + 5% CO2 3.3 2.4Peach slices 0 Air 3.0 3.0

0.5% O2 + 10% CO2 3.1 1.05 Air 5.5 4.6

0.5% O2 + 10% CO2 3.9 1.1Large muskmelon cubes 5 Air 4.0 4.0

1% O2 + 10% CO2 2.3 0.810 Air 9.6 10.6

1% O2 + 10% CO2 5.4 2.2Small muskmelon cubes 5 Air 2.7 3.8

1% O2 + 10% CO2 5.5 2.110 Air 5.2 7.0

1% O2 + 10% CO2 6.5 3.2Broccoli florets 0 Air 12.9 15.8

0.5% O2 + 10% CO2 6.9 8.15 Air 22.6 31.0

0.5% O2 + 10% CO2 7.7 11.310 Air 41.2 68.0

0.5% O2 + 10% CO2 15.3 20.5

Source: Extracted from Watada et al. (1996), Postharvest Biology and Technology, 9: 115–125, usedwith permission from Elsevier Science.

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Physiology of Fresh-cut Fruits and Vegetables 111

High CO2 atmospheres can have apparently contradictory effects on bacterialand fungal growth, depending on the product. A high CO2 atmosphere (30%) inhibitedboth phenylalanine ammonia lyase activity and accumulation of phenolics in shreddedcarrots, but lactic acid bacterial growth was rapid (Babic et al., 1993). The phenolicacids have been shown to be antibacterial in activity (Harding and Heale, 1980;Barber et al., 2000). This line of thinking is supported by the observation that lacticacid bacteria never grow in shredded carrots held in air atmosphere (Carlin et al.,1990). At the other end of the response spectrum is the well-known phenomena ofthe fungistatic effect of high CO2 atmospheres (Wells and Uota, 1970). Microbialgrowth is inhibited by commercial low O2, high CO2 atmosphere packages of minimallyprocessed iceberg lettuce (King et al., 1991). High CO2 (10%) showed suppressionof fungal and bacterial growth in fresh-cut honeydew, strawberries, and peaches (Qiand Watada, 1997). Low oxygen atmospheres (1 or 3%) had little effect on microbialgrowth in cut cantaloupe, but a combination of low O2 (3%) and high CO2 (7.5 or 15%)was effective in controlling microbial growth and decay in cut cantaloupe (Portelaet al., 1997).

CO2 can also have contrasting effects on quality retention in different products.High CO2 (15%) improved firmness retention in cut cantaloupe (Madrid andCantwell, 1993), whereas high CO2 caused tissue softening and electrolyte leakagein cut peppers (López-Gálvez et al., 1997a). Negative effects of CO2 on productquality may not be apparent until after product is removed to air atmospheres. HighCO2 (20%) increased extractable phenylalanine ammonia lyase (PAL) in cut lettucetissue, although phenolic accumulations and tissue browning are not a problem whilethe lettuce remained in the package (Mateos et al., 1993). This is due to the lowPAL activity in situ, because CO2 readily dissolves in the tissue and thereby reducescytoplasmic pH, which in turn, inhibits the activity of the enzyme. Once removedto air, the tissue pH rises and in situ PAL increases, providing substrates for browningreactions. This phenomenon was reported for butterhead lettuce in which browninghas been associated with high package CO2 levels, but the browning did not occuruntil the lettuce was transferred to air (Varoquaux et al., 1996b).

Low O2 (0.25%) reduces respiration, ethylene production, weight loss, browning,and microbial counts on sliced zucchini (Izumi et al., 1996). Low O2 has also beenfound to reduce browning in cut lettuce (López-Gálvez et al., 1997b; Smyth et al.,1998) and appears to reduce water loss in broccoli florets (Bastrash et al., 1993).Rapid development of low O2 and high CO2 is also key to controlling cut-surfacebrowning, and actively modified atmosphere packaging is commercially used inlettuce and potato products (Gorny, 1997).

One of the greatest challenges is that modified atmosphere packages cannotprovide acceptable O2 and CO2 concentrations for some commodities. Therefore, inmany cases, the attainable atmospheres are quite deviant from those considered optimal(Beaudry, 1999). While recommendations have been made for optimal atmospheresin which to package most fruit and vegetable products, the reality is that not allcommodities can be maintained in appropriate atmospheres using packaging films(Figures 5.13 and 5.14). For example, cut lettuce and lettuce salads are packaged inatmospheres that are very different than those recommended from prior researchreports (Figure 5.13) and that appear to do relatively well in commercial practice

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(López-Gálvez et al., 1997b). There are some risks associated with such packaging,but in practice, by using conservative shelf life expectations, these risks can be tolerated(Smyth et al., 1998).

CONSEQUENCES OF CUTTING-INDUCED INJURY ON QUALITY RETENTION

Wound-induced ethylene production is associated with an increased rate of ripeningin papaya flesh (Paull and Chen, 1997) and softening in pear and strawberry slices(Rosen and Kader, 1989). This leads to the conclusion that ethylene removal wouldbe beneficial to quality retention of packaged fresh-cut products. However, thebenefit of ethylene removal in modified atmosphere packages has yet to be demon-strated to be of significant merit. Ethylene removal has been shown to reduce soft-ening in kiwifruit and banana slices held in air atmospheres and yellowing of cutspinach, but ethylene removal was of no benefit in reducing yellowing of broccoliflorets (Abe and Watada, 1991). It has been pointed out that most fresh-cut packageatmospheres are highly modified and that high CO2 and low O2 would certainlyinhibit, if not completely prevent, ethylene action on the fresh-cut tissue (Gorny, 1997).This is supported by the work of Howard et al. (1994) who showed that ethyleneabsorption had little, if any, effect on the quality of diced onions held in modified

FIGURE 5.13 Recommended O2 and CO2 combinations for the storage of vegetables. Theshaded area depicts atmospheres theoretically attainable by modified atmosphere packaging(MAP) using low-density polyethylene (LDPE, lower boundary) and perforated packages (upper,dashed line). The darkened area represents atmospheres observed in commercial modifiedatmosphere packages of mixed, lettuce-based salads. (Reprinted from Beaudry 1999, PostharvestBiology and Technology, Vol. 15, pp. 293–303, with permission from Elsevier Science.)

VEGETABLES

MUSHROOMS

ASPARAGUS

OKRA

LDPE

TOMATOPEPPERARTICHOKERADISHLETTUCE

LEEKS

BROCCOLIBRUSSELSPROUTS

BEANSCABBAGE

PARSLEY

SPINACH

Attainableatmospheres

20

15

10

5

00 5 10 15 20

Oxygen (kPa)

Car

bon

Dio

xide

(kP

a)

Commercial pkgs. lettuce

Perforated packageCHICORYCELERYCAULIFLOWER

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Physiology of Fresh-cut Fruits and Vegetables 113

atmospheres (2.65% O2, 3.65% CO2 at equilibrium). In fact, they found increasedmicrobial growth in packages containing the ethylene adsorbent. This was likelydue to the fact that sulfur volatiles were also removed from the package headspaceby the absorbent, and it is known that some sulfur volatiles produced by injuredonion tissues have antimicrobial properties (Blanchard et al., 1996). No response orimprovement in quality was found with use of ethylene adsorbents in packaged,minimally processed carrot sticks (Howard and Griffin, 1993). Another issue regard-ing ethylene activity is that wounding induces the production of allyl isothiocyanatesin cabbage, and these compounds are strong antioxidants that can suppress bothwound-induced ethylene production and browning (Nagata, 1996). Therefore, accu-mulation of secondary metabolites may impinge on other physiological responsesto cutting, depending on the product in question, and thus, must be kept in mind.

Another aspect of wound-induced changes is the production of volatiles otherthan ethylene. Sulfur volatiles in onions are typically produced within hours of cutting,and their production may last for a few days (Toivonen, 1997a). If these volatilesare not removed from the tissue (i.e., with appropriate adsorbents), then physiologicaland biochemical changes can be initiated in response to their accumulation(Toivonen, 1997b) leading to declines in tissue quality. It is, therefore, very importantto minimize the effects of the responses to cutting in the early stages in order tocontrol quality changes. Also, it has been shown that production of some oxidation

FIGURE 5.14 Recommended O2 and CO2 combinations for the storage of fruit. The shadedarea depicts atmospheres theoretically attainable by modified atmosphere packaging by filmpermeation alone (low-density polyethylene, LDPE, lower boundary) and via perforations alone(upper, dashed line), or their combination (shaded area). (Reprinted from Beaudry 1999, Post-harvest Biology and Technology, Vol. 15, pp. 293–303, with permission from Elsevier Science.)

STRAWBERRYBLACKBERRYBLUEBERRYCHERRYFIG

PINEAPPLE

MANGOPAPAYA

PERSIMMON

TEMPERATECLIMACTERIC FRUITS

CRANBERRYPLUM

CITRUS

Attainableatmospheres

LDPE

GRAPE

OLIVE

FRUIT20

15

10

5

00 5 10 15 20

Oxygen (kPa)

Car

bon

Dio

xide

(kP

a)

Perforated package

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114 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

products of free fatty acids as a consequence of wound-induced membrane deteri-oration can lead to off-flavors (Rolle and Chism, 1987).

Storage temperature after cutting will certainly influence the effect of woundingon product quality. It was determined that quality of packaged tomato slices wasbetter maintained at 2°C than at 10°C. This was associated with the fact that ethyleneand respiration production rates were increased by cutting at the higher storage tem-perature as opposed to the lower storage temperature (Artés et al., 1999), despitethe fact that the atmospheres in the packages were less modified at the lower storagetemperature. In another example, honeydew cubes were firmer and had reduced res-piration and ethylene production in a 4% O2 and 10% CO2 atmosphere at 10°C,while there is no appreciable response seen at 5°C (Qi et al., 1999). This is likely dueto the fact that the lower temperature is already controlling quality deterioration,and the modified atmosphere can provide little additional benefits under those con-ditions. However, it must be cautioned that the quality of the honeydew cubes werepoorer (controlled atmosphere or not) at 10°C. Bacterial populations were lower incontrolled atmosphere conditions than in air at both 5 and 10°C.

The prediction of respiration rate is a very important factor in the selection ofthe appropriate packaging film for fresh-cut products (Lee et al., 1997). A plethoriaof information on the CO2 production by fresh-cut products exists (see Table 5.2),however, the oxygen level in the package is probably as important, if not more important,in the package selection. Avoidance of hypoxic or anaerobic conditions in the packageis considered to be critical to ensuring quality of fresh-cut product (Lakakul et al.,1999). Modeling of the ideal film for a product is relatively simple if the ideal temper-ature is assumed for storage and handling (Lee et al., 1997), however, in the realworld, most minimally processed products are exposed to non-ideal temperaturesduring distribution and retail (Talasila et al., 1995). Several research groups haveproposed modeling approaches to deal with non-ideal temperature situations. Manysophisticated approaches to model temperature dependence of respiration to assistin MAP film design have been published (Makino et al., 1997). Probably the mostmanageable is one that establishes that most abusive situations can be tested usinga single superoptimal temperature of 7°C (Jacxsens et al., 2000) and that gives areasonable selection for films usable in the range between 2 and 10°C. The greatestissue in obtaining accurate respiration values is which approach is used to determinerespiration rate at different atmospheres. There are two that are commonly practiced:use of flow-through respiration setups, which have been suggested to give the closestphysiologically correct values (Lee et al., 1997) and a closed or static respirationapproach, where respiration is assessed either in a semipermeable flexible film package(Lakakul et al., 1999) or in an impermeable chamber (Beveridge and Day, 1991; Gongand Corey, 1994; Jacxsens et al., 2000). The closed approach for respiratory behavioris likely a better simulation of a modified atmosphere package, because there is evi-dence that a flow-through system does not accurately duplicate actual quality changesin a package situation (Ben-Arie et al., 1991; Larsen and Watkins, 1995). Anotheraspect of the issue is that most package O2 and CO2 permeabilities change differentiallywith increases in temperature (Moyls et al., 1998; Lakakul et al., 1999). In some cases,CO2 transmission increases faster than O2 transmission (Lalakul et al., 1999), and inothers, the O2 transmission increases faster than CO2 transmission (Moyls et al., 1998).

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Physiology of Fresh-cut Fruits and Vegetables 115

TABLE 5.2Respiration Rates of Fresh-cut Fruits and Vegetables at 0–2.5°C and 10°C

Commodity Processed Form

Respiration (mg CO2 kg−1 h−1)

0–2.5°C 10°CGreen beans cut 7.1a 39.7Zucchini sliced 6.1a 23.9Cucumbers sliced 1.7a 4.9Yellow summer squash sliced 3.3a 9.0Bell pepper sliced 3.6a 7.1Tomatoes sliced 0.7a 5.1Kiwifruit sliced 3.7a 11.9Banana, without peel sliced 4.0a 10.7Strawberry hull removed 3.1a 11.3

sliced 8.5–11.2b —Green seedless grapes without stems 1.0a 2.9Peach sliced 3.1a 9.5Muskmelon (large type) cubed 1.9a 6.2Muskmelon (small type) cubed 1.4a 5.0Crenshaw melon cubed 0.6a 4.6Honeydew melon cubed 1.2a 4.2‘Anjou’ pears sliced 1.7–3.8a 6.4–7.5‘Bartlett’ pears sliced 2.8–4.9a 9.9–11.8‘Bosc’ pears sliced 1.7–2.7a 7.3–7.8‘Red Anjou’ pears sliced 1.7–4.4a 6.1–8.1‘Bintje’ potato whole, pre-peeled 3.0c 9.0

half 4.0c 12.0sliced 5.0c 20.0

‘Van Gogh’ potato whole, pre-peeled 4.0c 10.0half 4.0c 11.0

sliced 6.0c 20.0Red beet whole, pre-peeled 2.0c 10.0

cubed 5.0c 14.0grated 6.0c 20.0

Carrot, from muck soil whole, pre-peeled 3.0c 9.0sliced 6.0c 17.0grated 5.0c 20.0–25.0

Carrot, from sandy soil whole, pre-peeled 4.0c 11.0sliced 6.0c 15.0–18.0grated 8.0c 27.0

Onion whole, peeled 4.0c 11.0rings 7.0c 20.0grated 6.0c 12.0

Broccoli florets 12.9a 41.2Chinese cabbage half 5.0c 9.0

coarsely shredded 9.0c 25.0finely shredded 12.0c 30.0

(continued)

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Therefore, the specific transmission properties at different temperatures of the filmbeing considered must be ascertained to better predict the package atmosphereresponse at different handling temperatures.

The cutting process can induce several physiological responses that may interactin closed package systems. For instance, shredded cabbage may, depending oncultivar, produce significant levels of allyl isothiocyanates (Yano et al., 1986). Theaccumulation of these compounds has strong inhibitory effects on respiration,polyphenol oxidase activity, phenylalanine ammonia lyase activity, ethylene produc-tion, and ACC synthase (Nagata, 1996). Therefore, the totality of the physiologicalresponses must be understood to accurately predict the quality responses of packagedminimally processed fruits and vegetables. In addition, many fresh-cut products aremixes of various commodities. In the case of cut lettuce salads, would mixes con-taining shredded cabbage have less browning than mixes containing no cabbage?This is one of the next generation of questions that must be studied in packagedfresh-cut products.

CONCLUSION

The physiological effects of wounding are important factors in determining qualityand shelf life of most fresh-cut fruit and vegetable products. While respiration ratesare generally thought to be important in determining shelf life, there is no clearevidence that respiration rate changes in response to cutting are directly associatedwith deteriorative processes that lead to the end of useful shelf life. This may be becauseother processes are more limiting to shelf life than respiration. However, respirationrates of various fresh-cut products are important in selecting packaging films. It is

TABLE 5.2Respiration Rates of Fresh-cut Fruits and Vegetables at 0–2.5°C and 10°C (Continued)

Commodity Processed Form

Respiration (mg CO2 kg−1 h−1)

0–2.5°C 10°C‘Apex’ white cabbage quartered 4.0c 12.0

coarsely shredded 9.0c 25.0finely shredded 12.0c 30.0

‘Lennox’ white cabbage eighth sections 4.0c 10.0coarsely shredded 8.0c 22.0

finely shredded 9.0c 27.0‘Delicious’ apple sliced 2.4–3.5c —‘Golden Delicious’ apple sliced 2.9–5.1c —‘Fuji’ apple sliced — 3.0–8.5Iceberg lettuce shredded 3.9b 6.4

a0°C, b2.5°C, or c2.0°C.

Source: Adapted from Rosen and Kader (1989), Kim et al. (1993b), Mattila et al.(1994), Watada et al. (1996), Gil et al. (1998), Gorny et al. (2000).

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Physiology of Fresh-cut Fruits and Vegetables 117

surprising that respiration rates of various fresh-cut fruits and vegetable fall intothree or four ranges (Table 5.2). Therefore, if similar package atmospheres weredesired for all commodities, then only a few package film permeabilities would berequired for commercial purposes. Also, the mixing of different commodities toproduce salad mixes would be relatively straightforward, because most of the com-ponents might be expected to have similar film permeability requirements. However,if extreme packaging atmospheres are required, for example, those required to controlbrowning in iceberg lettuce (Figure 5.13) (Smyth et al., 1998), then special perme-ability requirements need to be specified. Even with such cases, it is important tohave information on the respiration rates of the product so that the appropriate filmcan be selected to avoid anaerobic package atmospheres.

The use of ethylene adsorbents to remove wound-induced ethylene may beredundant if atmospheres are modified (Gorny, 1997). Reduced O2 (<5%) and elevatedCO2 (>5%) will significantly inhibit ethylene effects. Rapid establishment of mod-ified atmospheres is especially beneficial in preventing the effects of softening anddeterioration produced by ethylene effects (Gorny, 1997). It may be that suppressionof ethylene action is one of the most important functions of modified atmospherepackaging in fresh-cut products, next to controlling water losses.

Wounding also results in a wide range of physiological effects on many aspectsof quality, including secondary metabolite accumulations, microbial growth, andwater loss. Appropriate cultivar selection, crop management, post-cutting treatments,and packaging can minimize these effects. It is the challenge of the commercialfresh-cut practitioner to integrate the physiological information that is available andcan be applied to their product and to determine the information that cannot beapplied directly. The challenge for researchers is to fill the existing gaps of knowledgeso that critical information is available to the fresh-cut industry.

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Wells, J.M. and Uota, M. 1970. “Germination and growth of five fungi in low-oxygen andhigh carbon dioxide atmospheres.” Phytopath. 60: 50–53.

Weston, L.A. and Barth, M.M. 1997. “Preharvest factors affecting postharvest quality ofvegetables.” HortScience 32: 812–816.

Wu, C.-M. and Liou, S.-E. 1986. “Effect of tissue disruption on volatile constituents of bellpeppers.” J. Agric. Food Chem. 34: 770–772.

Yano, M., Saijo, R., and Ota, Y. 1986. “Inhibition of browning and ethylene production inshredded cabbage by isothiocyanates.” J. Japan. Soc. Hort. Sci. 55: 194–198.

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Enzymatic Effectson Flavor and Texture of Fresh-cut Fruits and Vegetables

Olusola Lamikanra

CONTENTS

IntroductionLipoxygenase

Occurrence and FunctionEffect of Lipoxygenase on Senescence and Resistance MechanismLipoxygenase Mediated Aroma and Flavor CompoundsLipoxygenase and Phenolic Compounds Lipoxygenase in Fruits and Vegetables

AppleAsparagusBroccoli CucumberKiwifruitMelon StrawberryTomato

PeroxidasePeroxidase-Mediated Reactions Occurrence and Stability Effect of Peroxidase on Ripening and SenescenceEffect of Peroxidase on Plant Defense Responses Peroxidase-Catalyzed BrowningPeroxidase in Fruits and Vegetables

Apple AsparagusBroccoliCarrotCucumber

6

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MangoMelon OrangePeach PearStrawberryZucchini SquashOther Fruits and Vegetables

Polyphenol OxidaseOccurrence and DistributionPolyphenol Oxidase-Mediated Browning Reactions Effect of Polyphenol Oxidase on Plant Tissue Defense MechanismInhibition of Polyphenol Oxidase-Mediated Browning ReactionsPolyphenol Oxidase in Fruits and Vegetables

AppleCucumberLettuceMango48MushroomMelonPearPineapple Prunus Fruits

Pectic EnzymesSoftening of Fruits and Vegetables Polygalacturonase LyasesPectinesterasePectic Enzymes in Fruits and Vegetables

AppleKiwifruitMangoMelonPearPeach Tomato

ConclusionReferences

INTRODUCTION

Enzymes and substrates are normally located in different cellular compartments, andtheir transfer is actively regulated. Cutting of produce removes the natural protectionof the epidermis and destroys the internal compartmentation that keeps them sepa-rate. The first visually observed change at the cut surface of plant tissue is desiccation

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127

of the first layer of broken cells and one to a few additional subtending layers ofcells (Kolattukudy, 1984). Tissue disruption increases permeability and mixing ofenzymes and substrates that are otherwise sequestered within vacuoles. The conse-quent increase in enzymatic activity may cause sensory deterorations such as off-flavor, discoloration and loss of firmness. Tissues of fresh-cut fruits and vegetables,are thus, more perishable and senescence-prone, than the intact organs from whichthey are obtained. Rupture of membranes also triggers metabolic changes thatencompass wound healing processes (Picchioni et al., 1994; Ramamurthy et al.,1992; Bernards et al., 1999). This chapter is a review of the occurrence, distribution,function and properties of some enzymes that could significantly affect the flavorand texture of fresh-cut produce. The main enzymes covered are lipoxygenase,peroxidase, polyphenol oxidase and the pectic enzymes. Other related enzymaticreactions and conditions that potentially affect their activity in cut fruits and vege-tables are also discussed.

LIPOXYGENASE

O

CCURRENCE

AND

F

UNCTION

Lipoxygenase (linoleate: oxygen oxireductase–EC 1.13.11.12) (LOX) is present inmost plant tissues and, in the presence of oxygen, catalyzes oxidation of polyunsat-urated fatty acids (PUFA) containing a

cis,

cis

-1,4-pentadiene structure. The generallyaccepted nomenclature for the most common LOX isozymes is as follows: LOX I,which exhibits alkaline pH for maximal activity and isoelectric point of about 5.7;LOX II, with optimum pH activity and isoelectric points around 6.5 and 6.25, respec-tively; and LOX III, with a broad optimum activity pH centered around pH 7 andisoelectric point of 6.15 (Siedow, 1991; Boyes et al., 1992). While all three LOXtypes could be present in plants such as legume seeds (Siedow, 1991), some LOXtypes are more dominant in others, such as LOX II in kiwifruit (Boyes et al., 1992),asparagus (Gavanthorn and Powers, 1989), cucumber (Feussner and Kindl, 1994;Wardale and Lambert, 1980), tomato (Bonnet and Crouzet, 1977) and apple (Kimand Grosch, 1979). LOX I converts linoleic acid preferentially to 13-hydroperoxidederivatives, LOX 2 forms 9-hydroperoxide compounds while LOX III yields a mixtureof hydroperoxides. LOX types II and III, in the presence of fatty acids and dissolvedoxygen, co-oxidize carotenoids (Lebedev et al., 1978; Canfield and Valenzuela, 1993;Ueno et al., 1982) and chlorophyll (Cheour et al., 1992; Zhuang et al., 1994, 1997).LOX II is activated by calcium, whereas LOX III is inhibited by calcium (Siedow,1991). In apple flesh disks, fruits rich in calcium and/or phosphorous had lower LOXcontent (Marcelle, 1991). Kato et al. (1992) reported the appearance of new LOX(LOX IV, V and VI) in germinated soybean cotyledons. LOX V and VI preferentiallyproduced 13(

S

)-hydroperoxy-9-

cis

, 11-

trans

-octadecadienioc acid (13

S

-HPOD) as areaction product of linoleic acid, whereas LOX IV produced both 13

S

-HPOD and9(

S

)-hydroperoxy-10-

trans

, 12-

cis

-octadecadienoic acid. All three isozymes have pHoptima of 6.5, no activity at pH 9.0 and preferred linolenic to linoleic acid as substrate.

Conjugated hydroperoxy acids (HPO) produced by LOX catalysis undergometabolism by hydroperoxide lyase (HPO lyase). HPO lyase catalyzes the cleavage

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of HPO to aldehydes, such as

cis

-3-noneal and hexanal from linoleic acid HPO and

cis

-3-,

cis

-6-nonadienal and

cis

-3-hexenal from linolenic acid HPO (Grun et al.,1996; Wardale and Galliard, 1975). Besides hydroperoxidation, formation of oxoac-ids and ketodienes are catalyzed by LOX (Vick and Zimmerman, 1980; Sanz et al.,1993). Hydroperoxide isomerase could also catalyze the isomerization of hydroper-oxide as an intermediate reaction pathway (Vick and Zimmerman, 1976). HPO lyaseis a membrane-based/bound enzyme present in small amounts in plant tissue. Onthe basis of substrate specificity, HPO lyase has been classified into three types:9-HPO lyase, 13-HPO lyase and nonspecific HPO lyase (Perez et al., 1999a). Thesubstrate specificity determines aroma composition of many plant products, despitethe specific action of LOX. The formation of volatile aldehydes of chain lengths C6and C9 is widespread in fruits and vegetables (Kim and Grosch, 1981; Gray et al.,1999; Perez et al., 1999a) and proceeds rapidly when plant cells are disrupted in thepresence of oxygen. Siedow (1991) described two metabolic pathways for HPO(Figure 6.1). One involves the action of HPO lyase on the LOX product, 13-hydro-peroxylinolenic acid, resulting in the formation of hexanal and 12-oxo-

cis

-9-dodecenoicacid. The latter is then isomerized into the more stable 12-oxo-

trans

-10-dodecenoic acid,also known as traumatin or as “wound hormone.” Traumatin is known to mimic somephysiological effects associated with wounding in plant tissues, inducing cell divi-sion and subsequent callus formation. It can also be converted to the acid derivativeby a nonenzymatic oxidation of the aldehyde moiety. Traumatic acid also appears

FIGURE 6.1

The “lipoxygenase pathway” for the biosynthesis of jasmonic acid, traumatinand traumatic acid from the lipoxygenase product, 13-hydroperoxylic acid (Siedow, 1991).

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129

to be involved in plant responses to wounding. The second pathway is initiated bythe enzyme hydroperoxide cyclase (HPC) that catalyzes the formation of 8-[2-(

cis

-2’-pentenyl)-3-oxo-

cis

-4-cyclopentenyl] octanoic acid, commonly referred to as 12-oxo-phytodienoic acid. This compound serves as the precursor for the formation ofjasmonic acid that has also been suggested to be a promoter of plant senescence andto be involved with plant signal transduction.

E

FFECT

OF

L

IPOXYGENASE

ON

S

ENESCENCE

AND

R

ESISTANCE

M

ECHANISM

An increase in LOX activity is a common feature in senescent plant tissues. Thecatalysis of

cis,

cis

-1,4-pentadiene structures is related to the critical role of LOXin plant tissue senescence. Treatments believed to delay the onset of senescence,such as the addition of cytokinins or antioxidants reduce the level of endogenousLOX relative to untreated controls (Siedow, 1991). Inhibition of LOX delays ripeningand softening in peaches (Wu et al., 1999) and kiwifruit (Chen et al., 1999). LOXactivity has also been correlated with plant tissue development (McLeod and Poole,1994; Tanteeratarm et al., 1989), as well as pathogen (Avdiushko et al., 1993a;Gardner, 1991; Ohta et al., 1990) and insect (Avdiushko et al., 1997; Duffey andStout, 1996; Thaler et al., 1996) resistance mechanisms. The protective mechanismimplicated is the further catabolism of oxidation products to jasmonic acid andmethyl jasmonate, which are members of an intracellular signal transduction chaintransferring signal to the nucleus and selectively activating gene expression (Gardner,1991; Feussner and Kindl, 1994; Avdiushko et al., 1993a). An increase in LOXoccurs concurrently with a decrease in linolenic and linoleic acids and total fattyacids in cucumber plants inoculated with

Colletotrichum lagenarium

or tobacconecrosis virus (Avdiushko et al., 1993b). The involvement of LOX in disease resis-tance of kiwifruit is evidenced by the elevated LOX activity in

Botrytis cinerea

inoculated fruits (McLeod and Poole, 1994). Linoleic acid and LOX products oflinoleic acid also inhibit germination and growth of

Botrytis cinerea

in carrot slices(Hoffman and Heale, 1989). Gorst and Spiteller (1988) detected large quantities ofLOX-unsaturated fatty acid oxidation compounds, 10-hydroxy-octadeca-8,12-dienoic acid and 10-hydroxyoctadeca-8-enoic acid, in aqueous homogenates offreshly picked strawberries. These compounds possess fungi toxic activity and appearto be involved in the self-defense mechanism of the plant.

L

IPOXYGENASE

M

EDIATED

A

ROMA

AND

F

LAVOR

C

OMPOUNDS

Aroma compounds in fresh-cut fruits and vegetables and the involvement of LOXin flavor biogenesis are reviewed in Chapter 12. Flavor production by LOX pathwayis generally quiescent unless triggered by maceration or cell damage (Gardner, 1989).Free radical intermediates in the reaction damage biological membranes (Eskin et al.,1977), and a pool of free fatty acids results from lipid hydrolysis (Biale, 1975). Incauliflower florets (

Brassica oleracea

L., Botrytis group), LOX-mediated increasesin the lipid phosphate-to-protein ratio occur via free radical intermediates (Voisineet al., 1991). In envelope membranes from spinach (

Spinacia oleracea

L.) chloro-plasts, LOX catalyzes the rapid breakdown of fatty acid hydroperoxides (Blee and

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Joyard, 1996). Fatty acids are quantitatively the major precursors of volatile com-pounds responsible for the aroma of plant products. Typical flavors generated byLOX were demonstrated by treating LOX extracts from green sea algae (

Entero-morpha intestinalis

) with linolenic acid (Kuo et al., 1996). Fresh apple-like, green,cucumber, mango and algal aromas were found in the volatile concentrates. Theaddition of LOX to green bean puree also caused flavor and aroma changes describedas “unripe banana,” “grassy,” “straw” and “ammonia” (Williams et al., 1986). LOXcontributed to sourness in the puree. Lactones that are important flavors of peachesand apricots are produced via LOX activity (Crouzet et al., 1990; Sevenants andJennings, 1966). LOX pathway products from lettuce (

Letuca sativa

L.) are primarily

cis

-3-hexenal,

cis

-3-hexenol and

cis

-3-hexenyl-acetate (Charon et al., 1996). In bothgreen and red bell pepper homogenates, the addition of linoleic acid considerablyincreased the levels of hexanal and hexanol, whereas the levels of

cis

-3-hexenal and

trans

-2-hexenal are markedly enhanced by the addition of linolenic acid (Luning et al.,1995). In mushrooms (

Agaricus bisporus

), a clear relationship exists between theproduction of 1-octen-3-ol and LOX activity (Belinky et al., 1994).

L

IPOXYGENASE

AND

P

HENOLIC

C

OMPOUNDS

Phenolic compounds inhibit LOX activity and have the potential to prevent LOX-mediated oxidation of carotenoids in vegetables (Oszmianski and Lee, 1990). Cat-echin and epicatechin have the highest inhibitory potency, and

p

-coumaric and ferulicacids exhibit the lowest inhibitory efficacy. In general, flavans show the highestinhibitory effect followed by flavonols and acidic phenolic compounds. Thus, theconcentration of phenolics appears to have a high correlation with the prevention ofcarotene bleaching. The mechanism of LOX inhibition seems to be due to reductionof the free radical intermediates formed during lipoxygenation (Takahama, 1985).

L

IPOXYGENASE

IN

F

RUITS

AND

V

EGETABLES

Apple

Unsaturated straight-chain ester volatiles appear to arise in apples only by the LOXpathway (Rowan et al., 1999). An investigation of the biosynthesis of

R

-octane and

R

-5-

cis

octene-1,3-diol, two naturally occurring antimicrobial agents in apples andpears after application of labeled LOX substrates and LOX-derived metabolitesshowed that almost all precursors applied were partly transformed into

R

-octane-1,3-diol (Beuerle and Schwab, 1999). Linolenic acid derivatives, still containing the12,13-

cis

double bond and octanol derivatives oxy-functionalized at carbon 3 werethe most efficient precursors of the 1,3-diol. The relationship between mineralcontent, ethylene biosynthesis and LOX activity in ‘Jonagold’ apple is one in whichfruits rich in calcium and/or phosphorous have lower LOX activity, 1-aminocyclo-propane-1-carboxylic acid (ACC) content and ethylene emission, while fruits richin potassium and/or magnesium with relatively high K/Ca have a higher LOXactivity, ACC level and ethylene emission. The fruit mineral content is an agentacting on LOX activity and the ethylene-forming system with a consequent effecton storage capacity (Marcelle, 1991).

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Asparagus

Asparagus LOX utilizes only monolinolein and linoleic acid as substrates but exhibitslow activity with di- or tri-linolein substrates. The enzyme has a pH activity optimumof 5.5–6.0 and is stable at pH 4.5–8.0 when stored at 2

°

C (Ganthavorn and Powers,1989). Ganthavorn et al. (1991), based on the first-order kinetics nature of the heatinactivation, suggested that LOX isozymes in asparagus do not have different heatstabilities. However, Pizzocaro et al. (1988) observed a biphasic pattern of heat inac-tivation and a relatively heat stable LOX in asparagus compared to LOX extractedfrom beans, carrots and zucchini.

Broccoli

Temperature abuse during storage and handling of broccoli results in reduced poly-unsaturated fatty acids (PUFAs) content and LOX activity and increases thiobarbituricacid-reactive substances compared to those stored at 2

°

C (Zhuang et al., 1994, 1997).Treatment of broccoli heads dipped in hot water (45

°

C for 14 minutes), however,results in better retention of chlorophyll and soluble protein contents when storedat 20°C. This treatment also lowers LOX and chlorophyllase activities (Kazami et al.,1991). Optimal pH of broccoli floret LOX occurs at 5.5–6.0 (Zhuang et al., 1994).Modified atmosphere packaging (MAP) storage (7.5% CO

2

and 11.2% O

2

) of broccoliflorets increases chlorophyll (Chl) and reduces LOX-induced degradation of C-18PUFA (Zhuang et al., 1994). Changes in water-soluble LOX activity in MAP andnonpacked samples follow a trend similar to PUFA. LOX activity, C18 PUFA, C18PUFA hydroperoxides, total fatty acids, thiobarbituric acid-reactive substances(TBA-RS) and Chl also increase in senescent broccoli flower buds during postharveststorage of broccoli florets at 5

°

C. Lack of packaging causes significant decreases inmoisture, PUFA, LOX activity, Chl and soluble protein, and an increase in thiobar-bituric acid-reactive substances (TBA-RS) (Zhuang et al., 1995).

Cucumber

Free and bound polysomes contribute to LOX synthesis, and the isozymes are locatedin different compartments of the plant cell. The cucumber fruit is often used as a modelfor extracting and locating organelles (Feussner and Kindl, 1994; Wardale and Lambert,1980). In the determination of the intracellular location and organellar topology of LOXforms in lipid bodies, microsomes and cytostol, Feussner and Kindl (1994) demon-strated that LOX isozymes occur in different compartments of the plant cell. Thecatalytic properties of lipid-body and microsomal LOX in cucumber were also foundto be different. Optimum pH for lipid-body LOX was 8.5, whereas that of the micosomalLOX was 5.5. Wardale and Lambert (1980) observed high LOX activity in intactcucumber protoplasts from both the peel and flesh tissue of cucumber fruit. Fewer intactvacuoles and decreased LOX activity occurred following osmotic rupture than frommacerated tissue. The most active substrates for cucumber LOX are linoleic acid(100%), linolenic acid (77%) and arachidonic acid (23%) with optimum pH and tem-perature being 5.5 and 40

°

C, respectively. Calcium, EDTA, dithiothreitol, imidazole,cysteine and

P

-chloromercuribenzoic acid do not inhibit cucumber LOX activity.

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Kiwifruit

Kiwifruit LOX contains at least one class II LOX. The enzyme is relatively heatstable and exhibits higher activity relative to other fruits (Boyes et al., 1992). Themain fatty acids in ripening kiwifruit were palmitic (16:0), stearic (18:0), oleic (18:1),linoleic (18:2) and linolenic (18:3). LOX activity can be correlated with a decreasein firmness of fruits stored at 20

°

C. At 0

°

C, however, LOX is markedly inhibited toless than 30% of those in fruit stored at 20

°

C, and ripening is delayed (Chen et al.,1999). The pH profile of kiwifruit LOX is one in which two pH maxima (6.2 and7.6–8.0) occur when linoleic acid is the substrate. Linolenic acid causes a shift inpeak activities to 5.75 and 7.4–7.7. Maximum LOX activity of the fruit occurs at36–46

°

C.

Melon

Cantaloupe melon (

Cucumis melo

reticulatus

) shows no LOX activity in the middle-mesocarp (mesocarp) tissue of all maturities unlike in the hypodermal-mesocarp(hypodermis) (Lester, 1990). Linolenic and linoleic acid in hypodermis tissue andplasma membrane integrity also declined at 30 days post-anthesis. In the hypodermistissue, LOX appears to play a major role in cantaloupe melon senescence by theproduction of peroxides from free fatty acids, which in turn, results in the pertur-bation of membranes. Enzymatic activity increases with ripening and senescence.LOX activity during cantaloupe melon cell division and enlargement periods ofdevelopment is also higher than in the mature fruit. In hybrid honeydew (

Cucumismelo inodorus

Naud.), LOX activity occurred on both hypodermal- and middle-mesocarp tissues (Lester, 1998). The hypodermal-mesocarp tissues, however, showhigher activity than the middle-mesocarp tissues in the mature fruit. Such differencesin LOX activities could be attributed to differences in the development of the tissues andin tissue water deficit. Homogenizing the flesh of cantaloupe melon activates LOXand HPO lyase activity and alters the aroma profile appreciably (Wyllie et al., 1994).LOX-derived compounds such as

cis

-2-hexenol are produced concurrently with asubstantial loss of esters. Temperature and pH optima for LOX in cantaloupe are20

°

C and 7, respectively (Lester, 1990). Hybrid honeydew LOX has only one activitypeak at pH 6.8 and is temperature dependent with higher activities at higher tem-peratures. Increase in LOX activity that coincides with lipid biophysical changes inhybrid honeydew plasma membrane also occurs with a resulting loss in membraneintegrity (Lester, 1998).

Strawberry

LOX and HPO lyase in strawberry (

Fragaria

X ananassa Duch.) fruits are locatedmainly in the micosomal fraction (Perez et al., 1999a). Linolenic acid is the preferredsubstrate for strawberry LOX, forming 13- and 9-hydroperoxides in the proportion70:30. HPO lyase in strawberry cleaves 13-hydroperoxides of linoleic acid (13%relative activity) and linolenic acid (100% relative activity) to form hexanal and 3-

cis

-hexenal, respectively. A detrimental effect of ozone treatment on strawberry aromaoccurs with a reduced emission of volatile esters in ozonated fruits (Perez et al., 1999b).

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Tomato

LOX-induced conversion of linolenoyl and alpha-linolenoyl fatty acyl groups tohexanal and hexenal (

cis

-3-hexenal and

trans

-2-hexenal), respectively, during themaceration of tomato fruits greatly contributes to the mix of volatile compoundsthat determines tomato flavor (Gray et al., 1999). Formation of the C6 aldehydesfrom C18 polyunsaturated fatty acids (PUFAs) proceeds by way of a sequentialenzyme system involving LOX (that preferentially oxygenates at the 9-position)followed by a hydroperoxide cleavage system which is, however, specific for the13-hydroperoxy position (Gallard and Matthew, 1977). LOX and HPO lyase activ-ities determined in the microsomal fraction, a post-microsomal pellet (PMP) andcytosolic fraction of tomatoes indicated that the microsomal form of LOX had thegreatest increase in activity, with fruit development between the mature-green andbreaker stages (Riley et al., 1996). Activity declined as the fruit turned red, unlikeHPO lyase, in which the microsomal fraction activity dominated in all stages ofdevelopment. HPO lyase in tomato is insensitive to pH over the range 6–8 and doesnot show any significant change in activity with ripening. Soluble LOX activity alsopredominates in the PMP fraction of the fruit for all stages of development. For thesubcellular fractions, LOX activity is higher at pH 6 than at pH 7 and pH 8 at allstages of development (Riley et al., 1996).

PEROXIDASE

Peroxidases (POD; EC 1.11.1.7) are iron-porphyrin organic catalysts that are widelydistributed in plants and seem to be normal components of most plant cells. Mostflavor changes in raw and unblanched fruits and vegetables could be correlated toPOD activity, and there is an empirical relationship between residual peroxidaseactivity, off-flavors and off-odors in foods (Burnette, 1977; Loaiza-Velarde et al.,1997; Cano et al., 1998). Changes that occur in POD activity in wounded and fresh-cut fruits and vegetables significantly contribute to their product quality (Svalheimand Robertsen, 1990; Loaiza-Velarde et al., 1997; Lamikanra and Watson, 2001).

P

EROXIDASE

-M

EDIATED

REACTIONS

Reactions catalyzed by POD enzymes are typically peroxidatic, oxidatic, catalaticand hydroxylation (Vamos-Vigyazo, 1981). The peroxidatic reaction is the mostimportant. The mechanism of reaction essentially involves an oxidative action byway of an initial formation of a complex intermediate with a hydrogen acceptor.The transfer of hydrogen from a donor substrate results in a second complex inter-mediate before the regeneration of the POD enzyme and formation of a reactionproduct. The reaction can be summarized as follows:

ROOH + AH2 → H2O + ROH + A

ROOH can be HOOH or some other organic peroxide, AH2 is the hydrogen donorin the reduced form, and A is the hydrogen donor in the oxidized form. The hydrogendonor complexes and two univalent oxidation intermediate steps occur:

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134 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

POD + H2O2 → Complex I

Complex I + AH2 → Complex II + AH

Complex II + AH → POD + A

Peroxidative reactions could take place by way of a number of reaction inter-mediates. Although the enzymatic action appears to be capable of proceeding byway of heterolytic pathways (Dixon and Webb, 1964), peroxidase activity could becatalyzed by free radicals and retarded by radical scavengers (Adak et al., 1997).POD enzymes may also enhance radical scavenging properties (Staehelin et al.,1992; Wang, 1995). The optimum pH and temperature for peroxidase activity dependon the source of the enzyme. Optimum pH could range from 1.8–8.5 (Lee and Tu,1995; Rodrigo et al., 1996; Otter and Polle, 1997). Temperature optimum rangeappears to be 25–60°C (Koshiba and Matsuyama, 1993; Campos et al., 1996), butthere are cases reported in which peroxidase activity increased under freezing con-ditions (Scott, 1975; Cano et al., 1998).

OCCURRENCE AND STABILITY

Peroxidases occur in both soluble and bound forms in fruits and vegetables (Sanchezet al., 1993; Brzyska et al., 1992; DaSilva et al., 1990; Ingham et al., 1998). The PODisoelectric points that vary from 3.5–9.5 evidence the existence of both cationic andanionic forms of POD in plants (McLellan and Robinson, 1983). Bound POD areusually attached by ionic interactions to the cell wall in the intact cell (Giordani, 1977;Thompson et al., 1998), and possibly other organelles such as mitochondria andribosomes (Hideg et al., 1991; Raa, 1973). Properties of bound POD differ from thoseof POD isolated from the soluble fraction of the cell (Penon et al., 1970). POD enzymesin vegetables usually have high stability to heat and, because of this, they are oftenused as the index of effective heat treatment in processed vegetables. Heat stabilityand regeneration properties of POD are often influenced by whether they are in thesoluble or bound state. Ionically bound POD is more heat stable in mango andorange (Khan and Robinson, 1993; McLellan and Robinson, 1984) in contrast withthe findings for apple POD (Moulding et al., 1987). In pear, however, both the solubleand ionically bound POD are heat labile with only approximately 2% of the originalenzymatic activity remaining after 2 min at 80°C (Moulding et al., 1989). Heatinactivation of peroxidase is nonlinear. A large decrease in activity is observed duringthe initial stages of a given heat process, but the rate of inactivation then changes toa much slower process (Clemente, 1998; Powers et al., 1984). The biphasic nature ofheat inactivation of POD is related to the presence of isozymes with differing heatstabilities in vegetables (Yamamoto et al., 1962). Regeneration of POD following heatinactivation of enzymes from Brussels sprouts and cabbage (McLellan and Robinson,1981), apple (Moulding et al., 1987), kiwifruit (Prestamo, 1989) and mango (Khan andRobinson, 1993) occurs. POD was completely inactivated after 2–10 min heat treatmentof cauliflower at 80–110°C (Bottcher, 1975; Vamos-Vigyazo, 1981), and it takes only15 min to reduce its POD activity by 98% at 50°C (Lee et al., 1984). A heat-resistant

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isozyme in cauliflower that is responsible for less than 5% of its POD activity, however,takes over 30 min to reduce activity to 50% at 50°C (Lee and Pennesi, 1984). Regen-eration following heat inactivation of Brussels sprouts was not observed for isolatedPOD isoenzymes (McLellan and Robinson, 1987a). Arroyo et al. (1999) did notinactivate POD in lettuce, tomato, asparagus, spinach, cauliflower and onion withcombined high-pressure and low-temperature treatments. Heat-shock treatments (rang-ing from 45°C for 120 sec to 55°C for 60 sec), however, caused a decrease of about30% in POD activity from iceberg letuce (Loaiza-Velarde et al., 1997).

The location and types of POD enzymes in plant tissues were described by Kvar-atskhelia et al. (1997). Plant PODs are classified into two types (Welinder, 1992).Ascorbate peroxidase (APX; class I), which is from the plant chloroplast and cystol, isdistinguished from classical plant POD (class II) isozymes by significant differences intheir primary structure. APX is an H2O2-scavenging POD that uses ascorbate as anelectron donor in plants and algae (Miyake and Asada, 1996; Asada, 1992). APX occursin two isoforms with respect to its cellular localization. One isoform of APX is localizedin chloroplasts and has been found in both a thylakoid (tAPX) form and a solubleform in the stroma (sAPX). tAPX scavenges the H2O2 that is photoproduced in thethylakoids. The other form of APX is localized in the cytosol, and its function seemsto be the scavenging of H2O2 produced in the cytosol (Miyake and Asada, 1996). Duringplant development, APX activity is regulated by ascorbate content. Wounding, as infresh-cut processing, generally leads to an increase in ascorbate biosynthesis and content(Diallinas et al., 1997; Oba et al., 1994). Classical, secretory plant POD that share50–95% sequence homology within the superfamily of POD (class II) are assigned toclass III (Welinder, 1992). Because guaiacol (2-methoxyphenol) is commonly used asa reducing substrate, these enzymes are also referred to as guaiacol-type POD. Mostsecretory plant POD are glycosylated (Johansson et al., 1992). Unlike guaiacol PODthat is characterized by broad specificity with respect to electron donors and participatesin many physiological processes, such as the biosynthesis of lignin and ethylene,ascorbate POD exhibits high sensitivity for ascorbate as the electron donor (Asada,1992; Amako et al., 1994) and is specific in its physiological role in scavengingpotentially harmful H2O2 (Dalton et al., 1998) and free radicals (Wang, 1995).

In the presence of flavonoids, guaiacol POD can participate in scavenging H2O2

both in vivo and in vitro by catalyzing the H2O2 dependent oxidation of flavonoids.In the flavonol-guaiacol POD reaction, ascorbate had the potential to regenerateflavonols by reducing the oxidized product (Yamasaki et al., 1997). The sensitivityof ascorbate POD to thiol reagents (Chen and Asada, 1992) and p-chloromercuriben-zoate (Amako et al., 1994) distinguishes it from guaiacol POD. Complex I formedby APX in a POD peroxidatic reaction pathway is extremely unstable and is rapidlyconverted to complex II without the addition of a reducing substrate (Patterson et al.,1995), whereas guaiacol POD are characterized by stable complex I.

EFFECT OF PEROXIDASE ON RIPENING AND SENESCENCE

An important function of POD is related to its role in indole acetic acid (IAA) oxidationaction, by which it participates in growth regulation (Acosta et al., 1986; Grambow,1986). Peroxidases are considered to be indices of ripening and senescence (Haard, 1973;

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Scott, 1975; Vamos-Vigyazo et al., 1981). The induction of POD isoenzymes duringethylene-induced senescence is a common response in cultivars of C. sativus, otherspecies of Cucumis and other genera of Cucurbitaceae (Abeles et al., 1989). In fruitsof climacteric ripening, POD and IAA oxidase isozymes were reinforced with pro-gressing maturity, while in nonclimacteric fruits, where ethylene did not noticeablychange during ripening, only IAA oxidase isozyme concentration increased, whilethe POD isoenzyme concentration decreased (Vamos-Vigyazo, 1981). In developingpeaches, two POD activity peaks corresponded to the two periods of weight gain(Flurkey and Jen, 1978). The role played by POD in the cell wall degradation oftomato was demonstrated by Araujo (1985). Application of POD substrates includingIAA, NADH, KI and Na2SO3 led to an increase in weight loss of ripening tomatotissue. 7-Hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran inhibited tissue degradation.POD activity increased and IAA content decreased during development and senes-cence of Chinese cabbage, cabbage and spinach (Zhang and Zong, 1988). In man-goes, POD activity increased in pulp tissues up to the climacteric stage of ripening.In a parallel way, the quantity of extractable proteins decreased from preclimactericto ripe or senescent fruits. Changes in the three POD isozymes appear to be relatedto the ripening process (Marin and Cano, 1992). The activities of POD and chloro-phyllase in the peel of the ripe green mango fruit were also about half of the ripeyellow fruit (Ketsa et al., 1999). Changes in POD activities corresponded to theclimacteric and the onset of senescence in ‘Golden Delicious’ apples (Gorin andHeidema, 1976). Maximum specific POD activities also occur at the “small green”and “large green” ripening stages of strawberry (Civello et al., 1995). Kinetin, acytokinin involved in respiration and ethylene production, however, had no catalyticeffect on cantaloupe melon POD (Lamikanra and Watson, 2000). Guaiacol PODappears to be the main POD class involved in lignification of the cell wall, degra-dation of IAA, biosynthesis of ethylene, wound healing and defense against pathogens(Gazaryan et al., 1996; Kobayashi et al., 1996).

EFFECT OF PEROXIDASE ON PLANT DEFENSE RESPONSES

POD is thought to be important in a variety of plant defense responses againstpathogens. The involvement in POD plant defense mechanism is related to its role inthe shikimate intermediate pathway in the synthesis of aromatic amino acids, indoleacetic acid (auxins), cinnamic acids (precursors of phenylpropanoid phytoalexins),coumarins and lignins (Biles and Martyn, 1993). Other enzymes involved in theshikimate pathway, such as PAL, chalcone isomerase, polyphenol oxidase and shiki-mate dehydrogenase, are also associated with plant defense mechanisms or senescence.POD activity increased more rapidly in resistant than susceptible cucumber hypoco-tyls after inoculation with the pathogens Clasdoporium cucumerinum Ellis and Arth.(Svalheim and Robertsen, 1990) and Colletotrichum lagenarium (Pass.) Ell. andHals. (Hammerschmidt and Kue, 1982). The pattern of isozymes that was induced byfungal infection or resistant hypocotyls was similar to the pattern of isozymes inducedby wounding. POD enzymes from watermelon, muskmelon and cucumber inducedwith C. langenarium were found to be antigenically and electrophoretically similar(Smith and Hammerschmidt, 1988). However, based on the inability of ethylene toinduce resistance, and the inability of silver thiosulfate to increase susceptibility, Biles

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 137

et al. (1990) concluded that POD does not appear to play a role in limiting diseasedevelopment in the cucumber-C. lagenarium system, although ethylene action stillappeared to be necessary for lesion development and senescence. Pathogen andwound-induced POD activity have been demonstrated in fruits and vegetables (Bilesand Martyn, 1993; Mohan et al., 1993; Madi and Katan, 1998; Mayer and Harel,1979; Miyazawa et al., 1998; Moran, 1998; Sutha et al., 1996). Storage stabilityappears to be related to POD activity in cut vegetables. Baardseth and Slinde (1980)reported a correlation between shelf lives of cut vegetables and their POD andcatalase (CAT) activities. Swede (Brassica napus L.), which has long storage stabilityin the cut state, has a high activity of POD and CAT relative to cut carrot withmedium POD and CAT activity. Activity of POD and CAT were relatively low inBrussels sprouts. In addition to the relative activities of the enzymes in these vege-tables, swede also contains the highest amount of ascorbic acid per milligram ofprotein. Ascorbic acid, acting as a reducing agent and endogenous substrate to therespiratory chains that give ATP, could also contribute significantly to the stabilityof cut swede. Increased POD activity has also been correlated with lignification,pathogenisis-related proteins, hydroxyproline-rich glycoproteins and suberization(Biles and Martyn, 1993). POD activity, particularly APX activity, could be indicativeof oxidative stress in plant tissues (Gueta-Dahan et al., 1997; Kampfenkel et al., 1995).POD activities in cantaloupe melon, strawberry and lettuce were also suggested tobe indicative of the relative levels of oxidative stress in the fresh-cut products(Lamikanra and Watson, 2000).

PEROXIDASE-CATALYZED BROWNING

The ability of POD to contribute to enzymatic browning is related to its affinity toaccept a wide range of hydrogen donors, such as polyphenols (Richard-Forget andGauillard, 1997). They are able to oxidize catechins (Lopez-Serrano and Ros-Bacelo,1997), hydoxycinnamic acid derivatives and flavans (Robinson, 1991; Nicholas et al.,1994) and flavonoids (Miller and Schreier, 1985; Richard and Nicolas, 1989; Richard-Forget and Gauillard, 1997). Two possible mechanisms are proposed for POD-catalyzed browning reactions. One involves the generation of H2O2 during the oxidationof some phenolic compounds that is used as in a normal peroxidatic action to furtheroxidize the phenol, while the second involves the use of quinonic forms as substrateby POD. Both mechanisms indicate that the presence of polyphenol oxidase enzyme(EC 1.14.18.1) would enhance POD-mediated browning reactions (Richard-Forgetand Gauillard, 1997). POD oxidizes (+) catechin at low H2O2 concentrations via theformation of complex I and complex II. In this reaction, (+) catechin is also an excellento-diphenol for complex II reduction (Lopez-Serrano and Ros-Barcelo, 1997).

PEROXIDASE IN FRUITS AND VEGETABLES

Apple

POD activity increases with ripeness of apples with a peak in activity around theclimacteric stage of ripening in the peel and pulp tissues (Prabha and Patwardhan,1986; Kumar and Goswami, 1986). Activity also increases during controlled atmo-sphere storage (Gorin and Heidema, 1976) and apparently during normal cold storage

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of apples, although variations occur with cultivar (Vamos-Vigyazo, 1981). The isozymepatterns differ among cultivars and could be used for cultivar differentiation (Barnes,1993; Manganaris and Alston, 1993).

Asparagus

Basic isozymes of isoelectric point (pI) greater than nine predominate in asparagusPOD. The more acidic fractions are found in the spear tip (Powers et al., 1984).Optimum activity for green asparagus POD occurred at pH 7. The enzyme regen-erated after being subjected to temperatures between 90 and 125°C for short periodsof time. The regenerated asparagus POD reached its maximum activity after beingstored six days at 25°C (Rodrigo et al., 1996, 1997). The basic and acidic fractionsare similar in heat stability, although the acidic fraction is more readily regeneratedafter heat treatment at 70°C (Powers et al., 1984).

Broccoli

Toivonen and Sweeney (1998) demonstrated the importance of antioxidant protectionoffered by POD and superoxide dismutase (SOD) to retention of green color inbroccoli. POD and SOD activities were approximately 30% higher in ‘Greenbelt,’a cultivar with a low yellowing susceptibility, than in the more susceptible ‘Emperor’cultivar. The ratio of superoxide dismutase to peroxidase activity was also lower in‘Greenbelt.’ POD and SOD are also involved in the enhanced lipid peroxidation incell membranes that occurs under drought stress. With the time and intensity ofdrought stress, activities of SOD and POD decreased during the first two days,increased during days two through six, then decreased during days six through eightof drought stress (Yang and Yang, 1998).

There are no differences in POD, ascorbate oxidase and texture between MAPpacked and nonpacked broccoli (Barth et al., 1993a). Reduced ascorbic acid retention,moisture content, total chlorophyll and color retention in broccoli are, however, greaterwhen packaged in an atmosphere containing 8% CO2 and 10% O2. Barth et al. (1993b)observed lower POD activity in broccoli spears packaged using a semipermeablepolymeric film and stored 96 h at 20°C relative to nonpackaged broccoli spears.Ascorbic acid, chlorophyll and moisture retention were also greater in packagedbroccoli. A 1.5% O2 atmosphere relative to normal air inhibits ionically bound PODactivity in iceberg lettuce (Ke and Saltveit, 1989a). Packaging of stored lettuceunder this atmosphere also inhibits ethylene and respiration, ethylene-induced russetspot development and PAL and indoleacetic acid (IAA) activities, and it reducessoluble phenolic content in the vegetable. Exposure of lettuce tissue to elevatedCO2 increased ionically bound insoluble POD and ionically bound IAA oxidaseactivity but reduced soluble IAA oxidase activity (Ke and Saltveit, 1989b). The useof a test strip ethanol biosensor composed of a chromatogen and immobilizedenzymes (POD and alcohol oxidase) to detect quality changes in improperly designedmodified atmosphere packages that result in low injurious O2 levels or temperatureabuse was reported by Smyth et al. (1999). The biosensor was effective in detectinglow O2 injury in MAP samples of cut broccoli, cauliflower, lettuce and shreddedcabbage packages.

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 139

Carrot

Surface discoloration on cut carrots occurs concurrently with increased levels ofsoluble phenolics, lignin POD, phenylalanine ammonia lyase (PAL) and syrin-galdazine oxidase (SOX) activities over time. Steam treatment and subsequent stor-age at 2°C retard surface discoloration by inhibiting phenylpropanoid metabolism.Soluble phenolic and isocoumarin production and lignin formation were inhibited,and POD, PAL and SOX were inactivated in treated cut carrots (Howard et al., 1994).In a study to determine the effects of an ethylene absorbent and storage at 2°C onsurface discoloration and phenolic changes in MAP carrot sticks, lignin formationalso occurred and corresponded with development of white discoloration in storage.Slicing stimulated PAL and POD activity, and both enzyme activities remainedelevated during storage (Howard and Griffin, 1993).

Cucumber

Anodic POD isozymes from cucumber fruit vary widely in their substrate specificitybut react preferentially with aromatic amines (Miller et al., 1990). In spinach,degradation of chlorophyll appears to be regulated through the peroxidase-hydrogenperoxide pathway, which opens the porphyrin ring, thus resulting in a colorlesscompound (Yamauchi and Watada, 1991). An anionic isozyme in cabbage with anisoelectric point (pI) of 3.7 and was relatively heat stable, while a cationic isozyme,pI 9.9, was more readily inactivated by heat (McLellan and Robinson, 1987b). Low-temperature storage reduces browning and PAL activity but has no effect on PODactivity in shredded cabbage (Takahashi et al., 1996).

Mango

A number of POD isozymes have been extracted from mangoes. Khan and Robinson(1994) reported four POD isozymes—two anionic and the other two cationic. Theanionic isozymes have higher molecular weights of 40 and 44 kDa, respectively,than cationic isozymes with molecular weights of 22 and 27 kDa (Khan andRobinson, 1994). Marin and Cano (1992) found three isoenzymes of POD in theextracts from ‘Smith’ mangoes that moved toward the anode at pH 8.3. The isoen-zyme pattern of POD of ‘Lippens’ mango extracts was significantly different, show-ing four faster moving bands in addition to the three characteristic bands of ‘Smith’mango fruits. Pulp POD activity increases with degree of ripeness in mangoes (Marinand Cano, 1992; Prabha and Patwardhan, 1986).

Melon

Cantaloupe melon POD activity appears to be consistent with that of ascorbateperoxidase based on the ascorbate inhibitor when guaiacol is used as substrate andinhibition by β-mercaptoethanol, L-cysteine and p-chloromercuribenzoate (Lamikanraand Watson, 2000, 2001). The optimum activity temperature for cantaloupe melonenzyme was 50–55°C. The enzyme was stable at temperatures below 40°C and at50°C for up to 10 min. Over 90% of total activity was lost at 80°C within 5 min.The broad pH optima, 5.5–7.5 at 50°C and 6–7 at 30°C, are indicative of the presence

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of more than one POD isozyme in the fruit (Lamikanra and Watson, 2000). Twonative electrophoretic POD bands with approximate molecular weights of 240 and170 kDa, respectively, comprised of six acidic subunits (pI 5.1–6.1) were present incantaloupe (Lamikanra and Watson, 2001). POD activity in cut cantaloupe is inhib-ited by ascorbic acid. The inhibitory effect appears to be related to an intermediateascorbate-POD complex. The ascorbate-POD exhibits antioxidant properties that involvetrace metal ion cofactors. This enhanced antioxidant property protects β-carotene andextends product shelf life.

Honeydew melon and watermelon POD exhibit optimum activities at aroundpH 7 and 6.5, respectively. While the former has its maximum activity at 50°C, PODin watermelon is relatively more stable at higher temperatures than honeydew melon,pineapple and kiwifruit, and has an optimum activity temperature of about 70°C(Lamikanra and Watson, unpublished).

Orange

Orange contains a latent POD, which catalyzes the oxidation of a number of the fruit’sconstituents by H2O2. Mn++ was required for the activity of POD as an oxidase withO2 (Bruemmer et al., 1976). In the peroxidase H2O2 system, the most reactive constit-uent compounds were ascorbic, caffeic and gentisic acids. Quercetin was unreactivein this assay, unlike POD from some fruits and vegetables (Hemeda and Klein,1991). In the O2 and Mn++ system, ascorbic acid and catechol and p-phenylenediamine were unreactive. In orange juice, however, POD activity is very low, appar-ently because of the low H2O2 content of the fruit (Bruemmer et al., 1976). Cationicand anionic POD with pI values ranging between 4.5 and 9.0 and molecular weights22–44 kDa were also isolated from oranges (Clemente, 1998).

Peach

POD activity changes with the three stages of development in peaches. The solublePOD activity is highest in the mesocarp and the exocarp at stage II, and isoenzymaticchanges in these tissues corresponded to the transition from cationic isoenzymes,predominant at stage I, to anionic isoenzymes at stage III. The ionically boundperoxidase activity in these tissues is highest at stage I (Sanchez-Roldan et al., 1990).The endocarp of these drupes becomes lignified, while the mesocarp remains par-enchymatous with fruit development. Acidic POD from lignifying endocarp aresimilar to those of the fleshy mesocarp. The endocarp has a larger amount andnumber of basic POD than the mesocarp (Abeles and Biles, 1991).

Pear

The influence of harvest date on the occurrence of physiological disorders in pearsmight be related to the nonenzymic and enzymic systems responsible for catabolismof active oxygen species. Pears (cv. ‘Conference’) harvested seven days after theideal time for commercial harvest maintained a constant POD activity relative tothose harvested on time, but APX increased 2.5-fold. Concommitantly, the activityof SOD and CAT fell about fivefold and twofold, respectively, when the fruit was

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 141

picked more mature, indicating a higher potential for the accumulation of cytotoxicO2− and H2O2, respectively (Lentheric et al., 1999). POD activity is unevenly dis-tributed in the flesh of pear cultivars, and the degree of inhomogeneity differs ineach cultivar (Vamos-Vigyazo and Nadudvari-Markus, 1982). Localization of PODon the cellular particulate fraction is associated with sclereid development. Mineralnutrition in the fruit may profoundly alter lignin metabolism by affecting the local-ization and, hence, the metabolic control of POD (Ranadive and Haard, 1972). PODisozyme patterns in pear are cultivar specific, and this could be used for varietal identi-fication (Santamour and Demuth, 1980; Hudina et al., 1996). Pear POD is activelyinvolved in its enzymatic browning reactions (Richard-Forget and Gauillard, 1997),although the level of POD cannot be used as an indicator of browning potential(Vamos-Vigyazo and Nadudvari-Markus, 1982; Zhang et al., 1994).

Strawberry

Strawberry fruit POD activity decreases remarkably with fruit ripening, and theenzyme is found primarily in a membrane-bound form (Civello et al., 1995). Theenzyme shows low thermal stability, and maximum enzyme activity occurs at 30°Cand pH 6.0. Two basic POD isozymes of molecular masses 58.1 and 65.5 kDa weredetected at different maturities of the fruit by Civello et al. (1995). Lopez-Serranoand Ros-Barcelo (1995, 1996, 1997), however, found one basic POD isozyme of highisoelectric point in process-ripe strawberry fruit, which is the only component ofPOD polymorphism in the whole fruit. This isozyme is capable of oxidizing phenolsonly in the presence of H2O2 and lacks catecholase, cresolase and laccase activities.

Zucchini Squash

Acclimation to chilling temperature in squash may be involved in the activities ofPOD, along with a number of free radical scavenging enzymes. The developmentof chilling injury symptoms in zucchini squash (Cucurbita pepo L.) stored at 5°Cwas delayed by preconditioning the fruit at a temperature of 15°C for two days (Wang,1995). Temperature preconditioning treatment suppressed the increase in peroxidaseactivity and reduced the decline of CAT activity in squash during subsequent storageat 5°C. SOD activity also remained higher in temperature-conditioned squash thanin the untreated squash throughout storage. The activities of ascorbate free radicalreductase, ascorbate peroxidase and dehydroascorbate reductase increased initiallyafter storage for four to eight days and declined thereafter in control and precondi-tioned fruits, but enzyme activities increased to a greater extent and remained higherin preconditioned fruits than in the controls (Wang, 1996).

Other Fruits and Vegetables

POD isozymes in carrot, tomato, kiwifruit, cauliflower, green beans and horseradishare comprised of three and six isozymes, respectively, in carrot (36–70 kDa) andtomato (38–62 kDa), one in cauliflower (70 kDa), two in kiwifruit (45–43 kDa) anda range of isozymes (36–120 kDa) in horseradish (Prestamo and Manzano, 1993).Ascorbic acid inhibits POD activity in the extracts.

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POLYPHENOL OXIDASE

OCCURRENCE AND DISTRIBUTION

Polyphenol oxidase (PPO) is comprised of a group of copper protein complex enzymesthat catalyze the oxidation of phenolic compounds to produce brown pigments atcut or damaged surfaces of fruits and vegetables. In plants, PPO is predominantlylocated in the chloroplast thylakoid membranes (Sherman et al., 1991; Hind et al.,1995) and can exist in an active state or a latent state (Mayer and Harel, 1979). Theprotein precursor of the enzyme contains the targeting sequence necessary for importinto the chloroplast and insertion in the thylakoid membrane (Sommer et al., 1994).Lax and Cary (1995) reported that PPO is synthesized as precursor proteins havinga transit sequence for transport into the chloroplast but appear to lack sequences forspecific targeting into the thylakoid membrane. The distribution of PPO in differentparts of fruits and vegetables as well as ratios of particle-bound and soluble enzymeswith maturity vary considerably. Reports on the distribution of PPO in apples, forexample, appear to be inconsistent. Although present in all parts of the fruit, somereports (Harel et al., 1964; Stelzig et al., 1972) indicate substantially higher levelsin the peel than in the flesh, while others (Klein, 1987; Janovitz et al., 1989) foundhigher PPO in the cortex than in the peel. Murata et al. (1993) demonstrated thatPPO enzymes in five apple cultivars are mainly localized near the core and second-arily near the skin. During ripening, the concentration of particulate enzymesdecreased with the concurrent appearance of a soluble fraction. In mature applefruits, where vacuoles occupy most of the cells, PPO was detected immunochemi-cally near the cell walls with use of anti-apple PPO antibodies. In cells of immaturefruits and tissue culture, PPO was detected in organelles other than the vacuoles,presumably in plastids (Murata et al., 1997). Clarified juices from pear were prac-tically devoid of PPO activity, and PPO activity remained almost entirely in the pulp(Vamos-Vigyazo, 1981). In green leaves, PPO is predominantly located in the chlo-roplasts (Golbeck and Cammarata, 1981; Chazarra et al., 1999).

POLYPHENOL OXIDASE-MEDIATED BROWNING REACTIONS

PPO action usually results in the formation of highly reactive quinones that can thenreact with amino and sulfhydryl groups of proteins and enzymes as well as withother substrates, such as chlorogenic acid derivatives and flavonoids (catechins,anthocyanins, leucoanthocyanidins, flavonols and cinnamic acid derivatives). Thesesecondary reactions may bring about changes in physical, chemical and nutritionalcharacteristics and may also affect the sensory properties of fruits and vegetables.Quinones also contribute to the formation of brown pigments by participating inpolymerization and condensation reactions with proteins (Shahidi and Naczk, 1995;Mayer and Harel, 1979; Mathew and Parpia, 1971; Vamos-Vigyazo, 1981).

PPO enzymes are classified based on substrate specificity. They includemonophenol monooxygenase, cresolase or tyrosinase (EC 1.14.18.1), diphenol oxi-dase, catechol oxidase or diphenol oxygen oxireductase (EC 1.10.3.2) and laccaseor p-diphenol oxygen oxireductase (EC 1.10.3.1). Formation of o-quinones from

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monophenols, for example, proceeds by way of monophenolase (creolase) catalyzedhydroxylation to o-diphenols followed by diphenolase (catecholase) catalyzed oxi-dation of o-phenols to o-quinones (Figure 6.2). BH2 stands for an o-diphenolic com-pound. The first activity shows a lag period (Chazarra et al., 1999; Perez-Gilabertand Garcia-Carmona, 2000) that has been explained by taking into account the chem-ical steps of tyrosinase action that are necessary for the production of o-diphenol(Cabanes et al., 1987), whereas the catecholase activity shows no slow transitionphenomena (Perez-Gilabert and Garcia-Carmona, 2000). Depending on the phenol,o-quinones also show great differences in stability, and color intensities vary widelyfrom one phenol to the other (Lee and Jaworski, 1988; Rouet-Mayer et al., 1990).Action of PPO on substrates results in decrease of activity, some loss of copper fromthe active site of PPO, and modification of one or more of the histidine residues thatligand copper (Osuga and Whitaker, 1995). Peach, pear and banana PPOs do nothave creolase activity (Osuga and Whitaker, 1995). ο-Quinones may also contributeto PPO-accelerated degradation of anthocyanins into colorless products. The degra-dation mechanism involves PPO and catechol in a sequential process (Figure 6.3).Eggplant anthocyanins, for example, are rapidly degraded by phenolases in thepresence of catechin and chlorogenic acid (Sakamura et al., 1966). PPO extract fromstrawberry and D-catechin together also caused a 50–60% loss in anthocyaninpigments after 24 h at room temperature (Wesche-Ebeling and Montgomery, 1990).

FIGURE 6.2 (a) Oxidation of monophenol to diphenol and (b) oxidation of diphenol to aquinone (Whitaker and Lee, 1995).

FIGURE 6.3 Mechanism of catechol and phenolase induced degradation of anthocyanin(Shahidi and Naczk, 1995).

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Wounding and ethylene production induce PAL activities apparently by separatemechanisms (Abeles et al., 1992; Lopez-Galvez et al., 1996; Hyodo and Fujinami,1989; Ke and Saltveit, 1989a). PAL is the first committed enzyme and catalyzes therate-limiting step in phenylpropanoid metabolism that produces phenolase-catalyzedoxidizable substrates (Ke and Saltviet, 1989a; Martinez and Whitaker, 1995). PPOis activated as a result of disruption of cell integrity and when the content of theplasid and vacuole are mixed. Wounding apparently first induces an increase in PALactivity and, consequently, an increase in oxidizable substrates.

EFFECT OF POLYPHENOL OXIDASE ON PLANT TISSUE DEFENSE MECHANISM

Active PPO appears to be present in all photosynthetic organisms and performs someessential functions in plants, including deterrence of insects and fungal pathogens(Manibhushanrao et al., 1988; Hoagland, 1990; Sherman et al., 1995). The antisensedownregulation of constitutive and induced PPO expression results in hypersuscep-tibility to pathogens in tomato, suggesting a critical role for PPO-mediated phenolicoxidation in plant defense (Thipyapong and Steffens, 1997). Increased activities ofPPO, POD, LOX, chitinase and alpha-glucosidase were also detected in cucumberleaves in the vicinity of lesions caused by pathogens or phosphate application(Avdiushko et al., 1993b). Evidence of PPO involvement in the deterrence of patho-gens in other fruits and vegetables include apple (Sharma and Kaul, 1996), mush-room (Jolivet et al., 1998), pepper and eggplant (Ouf et al., 1998).

INHIBITION OF POLYPHENOL OXIDASE-MEDIATED BROWNING REACTIONS

Considerable research on the conditions under which PPO action is inhibited or retardedin food systems and on the compounds that have inhibitive effects on PPO activityhave been conducted. Inhibitors of PPO-catalyzed browning can act by way of acombination of reaction pathways. Some compounds prevent melanosis by directreducing action on PPO (Kahn and Andrawis, 1986; Sayavedra-Soto and Mongomery,1986), by reducing o-quinones to diphenols (Golan-Goldhirsh and Whitaker, 1984;Richard-Forget et al., 1992) or Cu2+ to Cu+ (Hsu et al., 1988) by interaction with theformation of o-quinone products (Embs and Markakis, 1965; Sanchez-Ferrer et al.,1989) or by decreasing uptake of O2 for the reaction (Embs and Markakis, 1965; Chenet al., 1991a). A number of PPO-catalyzed reaction inhibitors can act by more than onemechanism. Thiol compounds, for example, can act by reducing the active PPO siteCu2+ to Cu+ that is more readily lost from PPO (Osuga and Whitaker, 1995), directinhibitory actions on PPO (Dudley and Hotchkiss, 1989; Robert et al., 1996), reductionof o-quinone (Kahn, 1985; Richard-Forget et al., 1992) or by complexing with quinonesto form colorless compounds (Richard et al., 1991; Richard-Forget et al., 1992). Ascor-bate acts as a less-effective reducing agent than cysteine (Janovitz-Klapp et al., 1990)and converts o-quinones back to their corresponding o-diphenols. It also inactivatesPPO directly and, in the presence of low levels of copper, can aerobically destroyhistidine residues of PPO, releasing copper by a free radical mechanism (Osuga andWhitaker, 1995). Sodium erythorbate shows no inhibitory effect on PPO, but its

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 145

prevention of browning is due to reduction of the quinones formed in the enzymaticreaction back to original phenolic compounds (Gopalan et al., 1999). Benzoic acid,kojic acid [5-hydroxy-2-(hydroxymethyl)-γ-pyrone] and carboxylic acids of thecinnamic series have also been demonstrated to show inhibitory effects on PPO infruits and vegetables (Vamos-Vigyazo, 1981; Chen et al., 1991b).

The inhibitory effect of calcium appears to be related to its preservation of mem-brane structure or direct inhibition of PPO. Stem browning in lettuce can be reducedby washing stem disks with solutions of 0.3 M calcium chloride, 0.1 mM 2,4-dichlorophenoxyacetic acid (2,4-D) or 0.5 M acetic acid (Thomas-Barberan et al.,1997). Calcium also caused a 60% decrease in PAL activity but had no substantialeffect on the accumulation of phenolic compounds. Unlike acetic acid that irreversiblyinhibited PAL activity, 2,4-D had a 60% inhibition. It also had an inhibitive effecton the biosynthesis of phenolic compounds. A mixture of vinegar and acetic acidsolution could also be used to prevent lettuce butt discoloration (Castaner et al.,1996). Cysteine, resorcinol, EDTA and citric acid are also effective in the preventionof lettuce browning during storage.

Kahn (1985) demonstrated the inability of casein hydrolyzate and bovine serumalbumin to inhibit mushroom (Agaricus bisporus) PPO activity. L-Lysine, glycine,L-histidine and L-phenylalanine (in increasing order of effectiveness) inhibited activ-ity by up to 60%; L-cysteine at 0.4 mM gave full inhibition. Glycine, diglycine andtriglycine (in increasing order) were effective in lowering the final level of coloredmelanin formed by the action of mushroom PPO on dehydroxyphenylalanine. Treatmentof mushrooms with 4-hexylresorcinol was reported to induce discoloration of mush-rooms. The combination of 4.5% sodium erythorbate, 0.1% cysteine HCl and 100 ppmEDTA, adjusted to pH 5.5, was quite effective in preventing browning (Sapers et al.,1995). Monophenolase and diphenolase activities of mushroom PPO are inhibited byagaritine [β-N-(γ -L(+)-glutamyl)-4-hydroxymethylphenylhydrazine]. The inhibitoryeffect is not as much as tropolone but is comparable with those of L-mimosine andbenzoic acid, and more potent than azeliac acid (Cabanes et al., 1996). Agaritine,an abundant and characteristic compound from the Agaricus genus also removes theenzymatically generated o-quinones. It could thus be suggested that agaritine playsa role in vivo in the endogenous regulation of mushroom PPO activity and o-quinoneconcentration (Espin et al., 1998a).

The following is a recent review (Beaulieu and Gorny, 2002) on the effectivenessof some anti-browning compounds on fresh-cut fruits. Ascorbic acid was found tobe more effective than erythorbic acid in preventing surface browning in ‘Winesap’and ‘Red Delicious’ apple plugs stored 24 h at room temperature, and 1% citric acidenhanced their effectiveness (Sapers and Zoilkowski, 1987). Browning was restrictedeffectively in stored (4.4°C) vacuum-packed carambola slices that were treated with1 or 2.5% citric acid plus 0.25% ascorbic acid (Weller et al., 1997). Ascorbic acid-2-phosphate and ascorbic acid-2-triphosphate treatments also decreased browningin ‘Red Delicious’ apple plugs for 24 hours at room temperature (Sapers et al.,1989). ‘Fuji’ apple slices treated with 2% ascorbic acid had no significant browningor loss of visual quality for up to 15 days when stored at 10°C in 0.25 kPa O2 (Gilet al., 1998). Calcium, in combination with ascorbic acid (both generally appliedas 1% dips), was highly effective in preventing discoloration of fresh-cut apples

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(Ponting et al., 1972) and pears (Gorny et al., 1998; Rosen and Kader, 1989).Browning was retarded in slightly under-ripe ‘Bartlett’ and ‘d’Anjou’ pears treatedwith sodium erythorbate, CaCl2 and 4-hexylresorcinol after 14 days of storage at4°C, however, fresh-cut ‘Bosc’ pears suffered severe browning irrespective of inhib-itor treatment (Sapers and Miller, 1998). A post-cutting dip with 0.01% 4-hexylre-sorcinol, 0.5% ascorbic acid and 1% calcium lactate extended shelf-life of ‘Anjou’,‘Bartlett’ and ‘Bosc’ pear slices for 15 to 30 days (Dong et al., 2000). ‘Red Delicious’apple slices treated with a combined anti-browning dip (4-hexylresorcinol, isoascor-bic acid, N-acetylcysteine and calcium propionate) and held at 5°C maintained visualquality for five weeks, yet microbial decay was evident after four weeks (Buta et al.,1999). Analyses of organic acids and the major sugars revealed that the slices treatedwith the combinations of anti-browning compounds retained higher levels of malicacid and had no deterioration in sugar levels at 5 and 10°C, indicating that higherquality was maintained during storage. Browning was significantly reduced in fresh-cut banana slices treated with citric acid (0.5 M) and N-acetylcysteine (0.05 M) thatwere stored at 5°C or 15°C for seven days, and no microbial decay was observedduring the seven-day storage (Moline et al., 1999). A combination of 0.5% carra-geenan and 0.5% citric acid also inhibited browning in stored diced ‘Granny Smith’and ‘Red Delicious’ apples for seven to nine days at 3°C (Tong and Hicks, 1991).When cysteine is used as an inhibitor of enzymatic browning on sliced apples (Walkerand Reddish, 1964) or pears (Sapers and Miller, 1998), pinkish-red off-colored com-pounds are formed due to phenol regeneration with deep color formation (Richard-Forget et al., 1992). If off-color formation can be prevented, cysteine may prove to bean effective replacement to bisulfites, because cysteine is a naturally occurring aminoacid that has GRAS status for use as a dough conditioner (Code of Federal Regu-lations 21:184.1271 and 21:184.1272). Browning was reduced for only one day at0°C in ‘Golden Delicious’ fresh-cut apples treated with 0.1% cysteine (Nicoli et al.,1994). Ineffectiveness of the cysteine treatment was attributed to oxidation in thepackage and was likely due to the low concentration applied. Recently, Gorny et al.(2000) reported that a post-cutting dip (pH 7) of 2% ascorbic acid, 1% calciumlactate, plus 0.5% (w/v) cysteine significantly extended shelf life of ‘Bartlett’ pearslices by inhibiting loss of slice firmness and preventing cut surface browning.Consumers could not distinguish between pear slices treated with the preservativesolution and control pear slices. After 10 days of storage in air at 0°C, 82% of consumersjudged treated pear slices to be acceptable in appearance, and 70% judged flavor tobe acceptable.

When used in combination with ascorbic acid, 4-hexylresorcinol has been reportedto be a very effective inhibitor of cut surface browning on many fresh-cut fruits, includingapples and pears especially (Dong et al., 2000; Luo and Barbosa-Cánovas, 1996, 1997;Moline et al., 1999; Monslave-Gonzalez et al., 1993; Sapers and Miller, 1998). Between1 and 7 ppm of residual 4-hexylresorcinol was necessary to prevent browning onfresh-cut pear slices, stored up to 14 days at 2–5°C (Dong et al., 2000). Although4-hexylresorcinol is effective in preventing cut surface browning in fresh-cut pearslices, it is not currently considered GRAS by the FDA and may not currently beused commercially on fresh-cut fruit products. However, 4-hexylresorcinol may alsoimpart an unacceptable off-flavor on fruit products.

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 147

POLYPHENOL OXIDASE IN FRUITS AND VEGETABLES

Apple

Hydroxycinnamic acid derivatives (caffeoyl, coumaroyl and feruloyl) are the mostimportant phenolics in various apple cultivars at maturity (Amiot et al., 1992). Applefruit are characterized by a predominance of chlorogenic acid or 5’caffeoylquinicacid. Flavan-3-ols (catechins and procyanidins) are the second most important. Therelative amounts of these compounds have been correlated with PPO-induced brown-ing in apples (Vamos-Vigyazo, 1981; Amiot et al., 1992). Phenolic compounds andpolyphenol oxidase (PPO) activity in the fruits of 11 apple cultivars, before and afterbruising, showed that the extent of browning closely correlated with the amount ofphenolics (hydroxycinnamic derivatives and flavan-3-ols) degraded. Maturity didnot appear to greatly influence the development of browning (Amiot et al., 1992).Prabha and Patwardhan (1986), however, noted that during ripening, PPO activityincreases in the peel tissue but decreases in the pulp.

Barrett et al. (1991) reported that shifts in subcellular location of PPO occurduring controlled atmosphere storage (CA) of ‘Delicious’ apples. These shiftsoccurred sooner in apples that were stored under high CO2 conditions (2.5–6% O2,8–12% CO2) than those stored under normal CA conditions (2% O2, 3% CO2). PPOfractions obtained by centrifugation at 4000 × g and 100,000 × g decreased, whilesoluble and 200 × g fractions increased with storage. Isozyme patterns of PPO alsovaried in different subcellular fractions.

Cucumber

PPO activity in cucumber fruit is present only in the skin, unlike the fruit’s PODactivity in that it is present in the skin and the flesh, although POD activity is higherin the skin. Both enzymes in cucumber exhibit pH optima near 7.0 but exhibitdifferent temperature optima and thermostabilities (Miller et al., 1990). PPO activitycorrelates with resistance to downy mildew (Yun et al., 1995) and Pseudoperono-spora cubensis in cucumber (Li et al., 1991).

Lettuce

Ethylene-induced PAL activity is a good predictor of fresh-cut lettuce storage life(Couture et al., 1993). PPO enzyme in lettuce with an estimated molecular weightof 56 kDa quickly oxidized chlorogenic acid (5-caffeoyl quinic acid) and (−)-epicatechin. The optimal pH of chlorogenic acid oxidase and (−)-epicatechin oxidaseactivities are 4.5 and 7.8, respectively, and both activities are stable in the pH range6–8 at 5°C for 20 hours (Fujita et al., 1991). Heimdal et al. (1994), however, foundthat PPO in photosynthetic and vascular tissues of lettuce had native molecularweights of approximately 150 and 136 kDa, respectively. The enzyme in vascularand photosynthetic tissues in lettuce was classified as a 1,2-benzenediol:oxygenoxidoreductase (EC 1.10.3.1). In addition, two PPO-active bands (40 and 46 kDa),possibly subunits of the enzyme, were found in vascular tissue only. The isoelectricpoint for lettuce PPO was 3.6. PPO-induced browning of lettuce can be correlated

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with the soluble phenolic content (Ke and Saltveit, 1989b). The photosynthetic tissuehas a higher phenolic content than the midrib tissue. For both types of tissue, anincrease in PPO activity occurs during storage. The photosynthetic tissue in lettuce,however, seems to be better adapted for fresh-cut processing (Castaner et al., 1999).

Controlled atmospheres containing air + 11% CO2 caused tissue injury and inducedPAL activity in iceberg lettuce midrib tissue. Injury symptoms included brown stain(browning of epidermal tissue) and sunken epidermal areas (pitting) a few millimetersin diameter. Pitting occurred in high CO2 atmospheres at 5°C with no increase inphenolic content, but browning did not develop until the tissue had been transferredto air at 25°C. Lettuce tissue exposed to 1.5% of O2 + 11% CO2 had reduced PALactivity and lower soluble phenolic content than lettuce exposed to air + 11% CO2 (Keand Saltveit, 1989b). The slight PPO activity inhibition by low O2 appears to be relatedto the inhibition of ethylene action, the attendant effects on phenolic metabolism andIAA oxidase activity (Ke and Saltveit, 1989a). An elevated CO2 environment inhibitscinnamic acid-4-hydroxylase activity in lettuce, presumably by inhibiting productionof phenolic compounds (Siriphanich and Kader, 1985). However, susceptibility toCO2 injury varies with cultivar. ‘Climax’ lettuce exhibited more severe CO2 injurysymptoms than ‘Salinas’ and ‘Winterhaven’ cultivars (Siriphanich and Kader, 1986).Moderate vacuum packaging (mvp) in 80 µm polyethylene inhibited enzymaticbrowning over 10-day storage at 5°C. When packaged in 80% O2, 20% CO2 (80/20),more browning occurred in SL3-bags (59 µm multilayer co-extruded film) than inpolyethylene bags. Endogenous ascorbic acid was ineffective as an antioxidant indelaying browning (Heimdal et al., 1995). The initial physiological attributes of eightlettuce cultivars (‘Calmar,’ ‘El Toro,’ ‘Sea Green,’ ‘Pacific,’ ‘Monterey,’ ‘Salinas 88,’‘Salinas 86-13,’ and ‘Nerone’) and three maturity stages (immature, mature andovermature) did not correlate with storage quality when processed lettuce was keptin air or air plus ethylene for 1–4 days prior to air storage at 2.5–20°C. However,ethylene-induced PPO and PAL activities and browning intensity significantly corre-lated with the final visual quality of the ethylene-treated, minimally processed lettuceafter 6–10 days of storage (Couture et al., 1993).

Mango

It appears that quality maintenance of fresh-cut mangoes is more related to particularcombinations of anti-browning agents rather than the modified atmosphere createdinside the package. Combinations of anti-browning agents resulted in a reductionof browning and deterioration of fresh-cut mangoes stored at 10°C and were moreeffective than those applied individually (Gonzalez-Aguilar et al., 2000). Treatmentscontaining 4-hexylresorcinol and potassium sorbate, or D-isoascorbic acid reducedchanges in color and microbial growth and did not affect sensory characteristics offresh-cut mangoes.

Mushroom

Mushrooms are subject to severe enzymatic browning during handling and storageand may not respond well to treatment with browning inhibitor dips. Washing and

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 149

application of dips greatly increase perishability, primarily as a result of waterabsorption during treatment that increases an internal environment favorable tobacterial growth (Sapers et al., 1995). Increased browning of mushrooms at higherstorage temperatures also occurs due to the direct effect of temperature on enzymeactivity. Catechol oxidase activity in button mushrooms at 15°C was almost twice thatat 10° and 5°C (Rai et al., 1989). The skin tissue has greater tyrosinase activity andhigher concentrations of protein and phenol than the flesh tissue (Burton et al., 1993).

Melon

Lamikanra and Watson (2001) determined that the cantaloupe melon’s PPO contentwas relatively weak compared with those in apple and lettuce. The fruit’s contentof oxidizable phenolic compounds was also negligible. This explains the lack ofsignificant browning reactions in cut cantaloupe melon (Lamikanra et al., 2000).

Pear

Reported pH optimum for PPO from pear range from 4.3–7.0 (Espin et al., 1997;Saddiq et al., 1994; Zhou and Feng, 1991; Haruta et al., 1999). The enzyme showsmonophenolase activity (Espin et al., 1997), and the presence of particle-bound PPOin pears is evidenced by the reduced browning in filtered and centrifuged pear juices(Sapers, 1992). The rate-limiting step in the monophenolase reaction mechanism inpears and apples appears to be a nucleophilic attack of the oxygen atom belongingto the hydroxyl group at the carbon atom in the 4-position on the copper atoms ofthe enzyme’s active site (Espin et al., 1998b). Catechol, 4-methyl catechol anddopamine are good pear PPO substrates, but monohydroxy substrates show noactivity (Siddiq et al., 1994). The enzyme could be assayed in a continuous specto-photometric method using p-hydroxyphenyl propionic acid (PHPPA) with 3-methyl-2-benzothiazolinone hydrazone (MBTH) (Espin et al., 1997).

Flesh browning induced by CO2 treatment was closely related to PPO and PALactivity in ‘Niitaka’ pear (Park, 1999). PPO, PAL and cinnamate 4-hydroxylase(CA4H) activity in the flesh were considerably higher in fruits stored in 2% O2 +2.5% or 5% CO2 than air or 2% O2 at 5°C, but there was no difference in theseenzyme activities at 0°C. Contents of total phenolics in flesh significantly increasedwith enriched CO2 atmospheres.

Pineapple

The major PPO isozyme from pineapple appears to be a tetramer of identical subunits,each with a molecular mass of 25 kDa. The amino acid sequence of this isozymeindicates the presence of a high content of glutamic acid, glycine and serine and a lowcontent of sulfur-containing amino acids. The PPO did not show creolase activity,and preferred substrates were diphenols. Ascorbic acid and L-cysteine were potentpineapple PPO inhibitors (Das et al., 1997).

A physiological disorder usually caused by chilling or gibberellic acid (GA-3)treatment of whole pineapple is the internal browning of fruit known as blackheart

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or endogenous brown spot. This is mainly due to the oxidation of phenolic compoundscatalyzed by PPO to form brown products. Low temperature or GA-3 markedlyincreases PPO and PAL activities and the content of catechol, chlorogenic acid andcaffeic acid (Zhou and Tan, 1992). The relationship between PPO content and fruitsize with susceptibility to internal browning in ‘Smooth Cayenne’ pineapple wasdemonstrated by Botrel et al. (1993). Fruits most susceptible to internal browningwere those in the second largest category (1500–1799 g) in weight and those showingthe highest PPO activity. The smallest fruits were the least susceptible. Fruits mostresistant to internal browning had a high content of phenolic compounds and highPOD activity. Heat treatment of fruit in an incubator (32–50°C for 24 hours) followedby storage in a cold room at 15°C decreases PPO, POD, ascorbic acid, acidity andspoilage rate. Dipping fruits in hot water (55°C for 10 minutes) followed by storageat 15°C also reduces the development of internal browning but induced loss offirmness and weight during the later stages of storage (Selvarajah et al., 1998).

Prunus Fruits

PPO from five Prunus species fruits (peach, apricot, almond, plum and cherry) showoptimum pH ranges from 4–5.5, and the enzymes do not occur in a latent form(Fraignier et al., 1995). Multiple active forms occur as a result of proteolysis of onemajor form. Under denaturing conditions, the main active and proteolyzed formsare, respectively, monomers of 63 and 43 kDa. Peach PPO enzyme acts primarilyon the orthodihydroxy configuration with optimum pH range of 4.0–6.0 (Reyes andLuh, 1960; Haruta et al., 1999). A correlation exists between peach genotypes forphenolic content and enzymatic browning (Gradziel and Wang, 1993). Four PPOisozymes with different heat stabilities were isolated from clingstone peach. Noneof the isozymes had monophenolase activity, and they varied in their specificity forseveral diphenols (Wong et al., 1971a). In a study to determine the effect of PPOinhibitors phloroglucinol and resorcinol on clingstone peach PPO-catalyzed oxida-tion of 4-methylcatechol, Wong et al. (1971b) determined that while PPO catalyzedthe formation of 4-methyl-o-quinone, it did not play a role in the reaction withphloroglucinol, resorcinol, d-catechin or orcinol. The reaction between these phenolsand 4-methyl-o-quinone to produce red-brown color takes place nonenzymatically.

Chlorogenic acid content decreased rapidly during enzymatic browning, but thesusceptibility to browning seemed to be more strongly correlated with the initialamount of flavan-3-ols in several apricot cultivars. Chlorogenic and neochlorogenicacids, (+)-catechin and (−)-epicatechin, and rutin (or quercetin-3-rutinoside) are themajor phenolic compounds in apricots (Radi et al., 1997).

PECTIC ENZYMES

SOFTENING OF FRUITS AND VEGETABLES

Pectic enzymes have received considerable attention regarding their involvement inripening and softening of cell wall components. Firmness retention is an importantquality parameter in fresh-cut fruit and vegetable products (Agar et al., 1999; Gorny

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et al., 1999; Ji and Gross, 1998; Senesi and Pastine, 1996). Pectic materials are madeup of chains of 1-4 linked D-galacturonic acid units that are usually esterified tovarying degrees with methanol. These enzymes can be cross-linked in various ways,and they exhibit a wide range of solubilities from the highly soluble, extensivelycross-linked molecules of native protopectin to readily soluble short, unbranchedchains of low molecular weight (El-Ashwah et al., 1977; Legentil et al., 1995;Martin-Cabrejas et al., 1995). Pectins are important components of the cell wall andmiddle lamella in higher plants. They are essentially linear α-1,4-galacturonan chainswith some esterified carboxyl groups. The chains are interrupted by linked α-1,2-rhamnose residues, and covalently bonded arabinose and galactose as α-1,5-arabinanand β-1,4-galactan side chains linked to rhamnose, respectively (Pressey, 1986). Thenature and relative amounts of branching in pectin vary from one source to the other(Fischer et al., 1994; Massiot et al., 1988; Hoff and Castro, 1969; Ross et al., 1993).Branching of the galacturonan typically occurs more in the cell walls than in themiddle lamella (Fischer et al., 1994; MacDougall et al., 1997; Knee et al., 1975).Free carboxylic acids in glacturonic acid are involved in intermolecular linkagesthat act as cross-bridges that influence cell wall strength. Calcium appears to beinvolved in forming intermolecular bridges by interaction with free carboxyl groupsof pectic acid polymers to form insoluble salts that form ionic linkages betweenpectin molecules (Kohn, 1969; McFeeters and Fleming, 1991). Pectin occurs in mostplant materials, particularly in young and fruit tissues, and is a major constituent ofthe cell wall. Pectin molecules are involved in cross-linking other polysaccharidesand proteins in the cell wall (Cutsem et al., 1993; Qi et al., 1995). The cells in fruitsand vegetables are connected through the middle lamella that is pectinous in nature.Pectin impacts firmness to plant organs by providing adhesion to juxtaposed cellwalls in the middle lamella (Chen et al., 1998a; Crooks and Grierson, 1983; Luzaet al., 1992; Rihouey et al., 1995).

One of the most obvious changes that occurs during the softening of fruits andvegetables is the progressive solubilization and depolymerization of pectic sub-stances (Aspinall, 1980; McNeil et al., 1984; Bacic et al., 1988). Calcium ions andpectic enzymes play important roles in the softening process. There are a numberof ways by which calcium ions could affect softening. Loss of calcium from themiddle lamella could reduce ionic linkages between pectin molecules (Femenia et al.,1998; Hong et al., 1995; Tandon et al., 1984), and changes in ionic strength, resultingfrom calcium loss, could reduce its regulatory activity on cell wall hydrolase(Almeida and Huber, 1999), reduce cell turgor (Stow, 1993; Mignani et al., 1995)and, consequently, its stabilizing properties on membranes (Shackel et al., 1991;Picchioni et al., 1995). Cell turgor regulates the tissue tension, affecting structuralfirmness. Enzymatic breakdown of pectins in tomatoes affected the relative amountsof calcium bound to the pericap, placenta and gel parenchyma (Burns and Pressey,1987). There are two principal types of enzymes responsible for pectin degradationin fruits and vegetables. These are depolymerases (polygalacturonase and pectic lyase)and pectinesterase, also known as pectase, pectinmethylesterase or pectinmethox-ylase. Depolymerizers hydrolyze the glycosidic bonds (hydrolases) or break themby β-elimination (lyases). Pectinesterase (EC 3.1.1.11) catalyzes the de-esterifica-tion of pectin.

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POLYGALACTURONASE

Poly-α-1,4-galacturonide glycanohydrolase, commonly referred to as polygalactur-onase (PG), hydrolyzes glucosidic linkages as shown in Figure 6.4. Polygalactur-onases are specific for de-esterified galactoronans. The rate and extent of hydrolysisare dependent on the degree of pectin esterification (Jain et al., 1990; Le-Cam et al.,1994). Polygalacturonases can be classified into endozymes that randomly cleaveglycosidic bonds of pectic acids and polygalacturonates within the molecule at theα-1,4 linkages and exozymes that catalyze stepwise hydrolysis of galacturonic acidfrom the nonreducing end of the chain. Endopolygalacturonases (EC 3.2.1.15) occurin fruit and filamentous fungi but not in yeast or bacteria (Baldwin and Pressey,1988; Hadfield et al., 1998; Kawano et al., 1999). Exopolygalacturonases (EC3.2.1.67) occur in fungi, bacteria and plants (Bartley and Knee, 1982; Downs et al.,1992; Konno et al., 1986; Tae et al., 1997). In general, endopolygalacturonase (endo-PG) enzymes account for most of the polygalacturonase activity in ripe fruit (Brady,1987; Downs et al., 1992; Nogata et al., 1993), although both endo- and exo-enzymesare active in some ripening fruits (Bartley and Knee, 1982; Nogata et al., 1993).Endo-PG appears to catalyze solubilization of pectins within the cells followed byfurther hydrolysis by exo-PG (Pressey and Avants, 1973, 1976). Some fruits thatsoften markedly during ripening (e.g., pears and freestone peaches) contain not onlyendo-PG but also exo-PG. Other fruits (e.g., apples and clingstone peaches) containonly exo-PG, consistent with slow softening characteristics. Low levels of exo-PGare found in many vegetative and storage tissues (Pressey, 1986).

FIGURE 6.4 Splitting of glycosidic bonds in pectin by hydrolysis (polygalacturonase) andby β-elimination (pectate lyase and pectin lyase) (Pilnik and Voragen, 1989).

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 153

LYASES

Poly(methoxygalacturonide) lyase (pectin lyase; EC 4.2.2.10) and pectate transeliminase (pectate lyase; EC 4.2.2.2) cleave the α-1,4-galacturonosidic bond bytrans elimination of hydrogen on carbon 5 of the galacturonic acid with the oxygenon the glycosidic bond. An unsaturated C-C bond is created between the 4- and5-positions of the galacturonic acid residue at the nonreducing end of the fragmentreleased (Figure 6.4). Glycosidic bonds in pectin are highly susceptible to thisreaction. Pectin lyase depolymerizes highly esterified carboxyl pectin by splittingglycosidic linkages next to methyl esterified carboxyl groups by β-elimination, whilepectate lyase attacks glycosidic linkages next to a free carboxyl group. Most lyasesare, however, specific for esterified galacturonans (Bruchlmann, 1995; Chen et al.,1998b). The enzymes are almost exclusively from microorganisms, although thereare indications of their natural occurrence in some fruits (Albersheim and Killias,1962; Medina-Escobar et al., 1997). Lyases produce unsaturated monomers thatrearrange to the 2-keto-uronic acid (Glover and Brady, 1995; Renard et al., 1991;Rong et al., 1994).

PECTINESTERASE

Pectinesterase (PE), also known as pectin pectyl-hydrolase, catalyzes the demethy-lation of esterified pectin (Figure 6.5). The enzyme specifically hydrolyzes themethylester groups of the C6 position of galacturonic acid and plays a crucial rolein the degradation of cell walls in higher plants, rendering highly polymerized pectinsusceptible to further degradation by PG (Fischer and Bennett, 1991). For example,in tomato, pretreatment of the wall with purified PE rendered walls from all ripeningstages equally susceptible to PG (Koch, 1989). Early ripening tomato varieties alsoshow higher PG and PE activity at all the stages, as compared to the late ripeningvarieties, where PG and PE activity increases during ripening (Young, 1995; Thakur and

FIGURE 6.5 Fragment of pectin molecule and points of attack by pectic enzymes (Pilnikand Voragen, 1989).

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Pandey, 1999). Unlike PG, PE is more commonly present in large amounts in unripefruit (Tucker and Grierson, 1982). PE can also prepare a substrate for pectate lyaseactivity. The enzyme is specific for polygalacturonide esters and will not hydrolyzenongalacturonide methyl esters or those in short-chain galacturonans to a large extent.They are activated by divalent or monovalent cations at high concentrations and havea pH optimum activity range of 5–8 (Pressey, 1977). The distribution of methoxygroups apparently affects the reaction rate of the enzyme. Methoxy groups adjacentto free carboxyl groups are removed at a more rapid rate than those next to esterifiedresidues on pectin molecules (MacMillian and Sheiman, 1974). PE activity presum-ably requires a free carboxyl group next to an esterified group on the galacturonidechain and proceeds linearly as methoxy groups are removed along the pectin chainuntil an obstruction is reached. Blocks of free carboxyl groups are thus produced asa result of PE action (Contreras-Esquivel et al., 1999; Grasdalen et al., 1996; Houand Chang, 1997; Kohn et al., 1985).

PECTIC ENZYMES IN FRUITS AND VEGETABLES

Apple

The physiologically active cell wall degrading enzymes in apple are PE, exo-PG,β-galactosidase and β-1,4 glucanase (Pollard, 1975; Knee, 1993; Abeles and Takeda,1990). Endo-PG is absent in the fruit. This makes the mechanism of ripening differentfrom fruits like peach and pear, in which the endo- and exo-PG are present in themature fruits. Softening of the cortical tissue of ripening apples is typically characterizedby the loss of galactose residues from the cell wall and an increase in soluble pectin.Apple exo-PG has a pH optimum of 4.5–5, is inhibited by EDTA and citrate and isactivated by Ca2+ and, to a lesser extent, by Sr2+. The enzyme which has a molecularweight of approximately 58 kDa, degrades apple cortical cell wall preparations, releas-ing low molecular weight uronic acid and polyuronide residues (Bartley, 1978). Soft-ening of apples during cold storage is accompanied by increased PG and Cx activities(Mahajan, 1994). On the tree, however, the process occurs concurrently with a decreasein Cx activity (Abeles and Takeda, 1990). Organic solvent extracts of apples inhibitβ-galactosidase (Dick et al., 1984; Lidseter et al., 1985). These compounds that inhibitβ-galactosidase have been identified to be chlorogenic acid, catechins and quercetinglycosides (Dick et al., 1985). β-Galactosidase in apple is believed to be responsible forthe loss of galactose residues that result from hydrolysis of the galactan of the primarycell wall. Soluble polyuronide is derived from the middle lamella region of the wall(Knee, 1993). Solubilization of polyuronides is the main degradative activity thathas been correlated with softening of apples (Bartley and Knee, 1982). β-Galactosidaseactivity increased, and cell wall galactose content decreased during softening of ‘Lodi,’‘McIntosh,’ ‘Golden Delicious’ and ‘York Imperial’ apples. The decrease in wall galac-tose was, however, least in ‘Lodi,’ which contained the lowest β-galactosidase activity.‘York Imperial,’ which softened most slowly, showed the highest β-galactosidaseactivity throughout storage (Wallner, 1978).

The lack of PG involvement as a determinant in the softening of apple has alsobeen suggested (Kang et al., 1999; Yoshioka et al., 1992). Kang et al. (1999) found

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no detectable PG during the softening of ‘Fuji’ and ‘Tsugaru’ apple cultivars. Solubleenzyme activities involved are in decreasing order α-mannosidase>β-arabinosidase>β-galactosidase>α-galactosidase during softening of ‘Fuji’ apples, while the activitiesare in the order of β-galactosidase>α-mannosidase>β-arabinosidase>α-galactosidaseduring softening of ‘Tsugaru’ apples. In particular, β-galactosidase activity increasedrapidly in both cultivars. Yoshioka et al. (1992) also detected no PG activity duringsoftening of apples and suggested de-esterification of polyuronides with a highdegree of methoxylation rather than depolymerization of polyuronides in the solu-bilization of polyuronides during ripening of the fruit. Irradiating apples (20 krd)accelerates pectin degradation and increases PE activity causing premature softeningof fruit (Flores et al., 1971).

Two forms of PE are present in apples. They differ both in charge and molecularweights. Their molecular weights are 55 and 28 kDa, respectively, and the heavierform is stable up to 40°C. Optimum activity is in the pH range 6.5–7.5 (Castaldoet al., 1989). Storage of apples at 38°C for four days and subsequent storage at 0°Cdecreased softening of ‘Golden Delicious’ apples in spite of comparable PE levelswith the unheated fruit, suggesting the lack of involvement of PE in apple ripeningand softening when preheated (Klein et al., 1995). Heat treatment also inhibited PGactivity but not that of PE. At the end of the ripening period, heated apples retainedmore insoluble pectin and were crisper than controls (Lurie and Klein, 1991).

Kiwifruit

Kiwifruit (Actinidia deliciosa) offers a useful alternative to tomato in which to studyfruit softening. Once harvested, there is an extended period during which most ofthe fruit softening occurs. Starch degradation and cell wall changes occur, includingpectin solubilization, loss of galactose from the pectin side chains, reduction inmolecular weight of the xyloglucan and cell wall swelling. Tensile tests and ultra-structural studies have shown that a loss of cell-cell adhesion at the middle lamellaoccurs toward the end of fruit softening (MacRae and Redgwell, 1992). Sutherlandet al. (1999) reported the predominance of pectin compared with cellulose alsooccurring at the middle lamella wedge near intercellular spaces of a number ofkiwifruit cultivars. Negatively charged groups and, to a lesser extent, galacturonicacid residues were preferentially located near the cell wall/plasma membrane bound-ary. Cellulose remained intact across the cell wall at all stages of fruit ripening,while distribution of xyloglucan was scattered throughout the wall later in ripening.

Mature kiwifruit shows a rapid drop in firmness after harvest to approximately2.5 kgf, after which softening slows down considerably. The outer pericarp softensmore rapidly than the core of the fruit (MacRae et al., 1989; Wegrzyn and MacRae,1992). Wang et al. (1995a) reported two stages of softening in ‘Qinmei’ kiwifruit.The first stage, the rapid softening phase, involves starch degradation, with amylaseas the key enzyme involved. Water insoluble pectin and cellulose apparently werereduced, and the activities of PG and Cx were markedly increased in the secondphase with no change in PE. Most cell wall galactose appears to be lost before pecticsolubilization during ripening (Redgwell and Percy, 1992; Redgwell and Harker,1995), apparently due to an endo-β-galactosidase enzyme system (Ross et al., 1993).

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Endo-PG activity increases during kiwifruit softening, but its action alone cannotexplain the pectin solubilization process (Redgwell et al., 1991). Miceli et al. (1995)found that cell wall changes during the softening of kiwifruit cv. Hayward areaccompanied by a decrease in the total structural polysaccharides due, in particular,to a reduction in the amounts of pectin and hemicellulose. Significant variations inthe quality were mainly due to an increase in highly methylated pectins and to aloss in protopectins. The amounts of arabinose, xylose and uronic acid in the cellwall polysaccharides also changed considerably during ripening (Miceli et al., 1995).

β-Galactosidase in ripening kiwifruit consists of several basic isoforms withmolecular weights ranging between 33 and 67 kDa. The optimum activity of theenzyme for p-nitrophenyl-β-D-galactopyranoside occurs at pH 3.2 but shifts to pH4.9 for a galactan purified from kiwifruit cell walls. The enzyme, which is specificfor galactosyl residues in the β-configuration, releases galactose from a variety ofkiwifruit cell-wall polysaccharide fractions, including cell wall material, Na2CO3-soluble pectin, high molecular weight galactan, xyloglucan and galactoglucomannan.It attacks the nonreducing end of galactose side chains, cleaving single galactoseresidues that may be attached to the 2, 3, 4 or 6 position of the aglycone (Ross et al.,1993). The loss of cell wall-associated galactose and pectin solubilization in kiwifruitcannot be correlated (Wegrzyn and MacRae, 1992; Redgwell and Harker, 1995).However, the ability of β-galactosidase to cause solubilization of pectin and decreasethe galactosyl residues of galactans associated with cellulose could partially explainhow fruits soften independent of PG activity (Gallego and Zarra, 1998).

Rapid softening of kiwifruit occurs in response to ethylene treatment that appearsto be initiated by an induction of pectinesterase activity, causing increased de-esterification of cell wall pectins, followed by degradation of solubilized pectin.Ethylene treatment also causes a slight increase in PG activity during fruit soft-ening, while β-galactosidase (EC 3.2.1.23) activity remains constant (Wegrzyn andMacRae, 1992).

Pectin solubilization, galactose loss and cell wall swelling that occur in kiwifruitare general features of cell wall changes that are not specific to postharvest ethylenetreatments (Redgwell and Percy, 1992). Application of CaCl2 reduces PG activity,leading to less degradation of protopectin, a smaller increase in water-soluble pectinand firmer fruits (Wang et al., 1995b; Xie et al., 1996).

Mango

Softening of mango causes an apparent overall loss of galactosyl and deoxyhexosylresidues, the latter indicating degradation of the pectin component of the cell wall.The loss of galactose appears to be restricted to the chelator-soluble fraction of thewall pectin, while loss of deoxyhexose seems to be more evenly distributed amongthe pectin regions. The chelator-soluble pectin fraction is progressively depolymer-ized and becomes more polydisperse (Muda et al., 1995). The inner mesocarp ofthe fruit is softer than the outer at each stage of maturity (Mitcham and McDonald,1992). Cell wall neutral sugars, particularly arabinosyl, rhamnosyl and galactosylresidues, decrease concurrently with a reduced size of hemicellulose and increasedPG activity during ripening (Mitcham and McDonald, 1992). β-Galactosidase activity

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also increases, while PE activity decreases continuously during softening (Abu-Sarraand Abu-Goukh, 1992; Ketsa et al., 1998). Abu-Sarra and Abu-Goukh (1992)reported decreases in PE activity of three mango cultivars (‘Kitchner,’ ‘Dr Knight’and ‘Abu-Samaka’) with ripening and were able to correlate decreased PE activitywith softening better than PG. The firmer mango cultivar ‘Keitt,’ exhibits moreloosely associated chelator-soluble pectin, accumulates more soluble polyuronidesand retaines more total pectin at the ripe stage than the less firm ‘Tommy Atkins’cultivar. Both cultivars have similar PG activity that increases with ripening. Theamount and molecular weight of cell wall hemicellulose decreased with ripening inboth cultivars (Mitcham and McDonald, 1992). Increased quantities of calcium arereleased into the fruit flesh as ripening progresses. This also contributes to loss oftexture and consequent softening (Tandon and Kalra, 1984).

β-Galactosidase apparently plays an important role in the softening of man-goes. Softening of ripening mango fruits was more closely related to changes inβ-galactosidase activity than to PG and PE activities (Ketsa et al., 1998). Fruit extractsof ripening ‘Harumanis’ mango contained a number of glycosidases and glycanases.Among the glycosidases, β-D-galactosidase appeared to be the most significant. Mangoβ-galactosidase has at least three isoforms: β-galactosidase I, II and III, with apparentKm values for p-nitrophenyl β-D-galactoside as 3.7, 3.3 and 2.7 mM. Optimum activ-ities are at pH 3.2 for β-galactosidase I and II and pH 3.6 for β-galactosidase III(Ali et al., 1995).

Cold storage (4°C) reduces softening of fruit. Chilled fruit contain higher levelsof ammonium oxalate-soluble pectin and less water- and alkali-soluble pectin thannonchilled fruit. Correspondingly, PG and β-galactosidase activities are reduced,and PE is increased compared to nonchilled fruit (Saichol et al., 1999). Roe andBruemmer (1981) reported an increase in both PG and Cx activities in mangoes storedat 4°C, and that Cx activity correlated better with softening than PG. PE activityalso decreased with time. In chilled ‘Keitt’ mangoes, water- and alkali-soluble pectindeclines, and ammonium oxalate-soluble pectin increases as the fruit becomes soft.The decline in alkali-soluble pectin, which takes place slowly in the cold fruit,correlates well with loss of firmness.

Melon

Loss of flesh firmness in cantaloupe melon (Cucumis melo reticulatus) occurs withmodifications of pectic and hemicellulosic polysaccharides, and a net loss of non-cellulosic neutral sugars. Increase in solubility and decrease in molecular size ofpolyuronides appear to be unrelated to PG activity (Gross and Sams, 1984; Lesterand Dunlap, 1985; McCollum et al., 1989). Luo et al. (1996), however, correlatedincrease in PG with ethylene production and fruit softening in the ‘Hetao’ muskmeloncultivar. Reduction in size of hemicelluloses is accompanied by increase in neutralsugar composition with xylose as the dominant sugar. Xylose also predominates inthe smaller polymers that are found in cantaloupe melon during ripening (McCollumet al., 1989). Glucose may, however, suppress PG production in vivo (Zhang et al.,1999a). Changes in several glycosidase activities in the mesocarp tissue take placewith ripening and softening (Fils-Lycaon and Buret, 1991; Ranwala et al., 1992).

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Glycosidase of melon mesocarp can be classified into three groups based on theirspecific activitiy patterns during fruit development and ripening. The first group (A)is composed of α-D-galactosidase, α-D-mannosidase and α-L-arabinofuranosidase.Their specific activities decrease during ripening. In the second group (B), β-D-galactosidase, β-D-glucosidase and α-L-arabinopyranosidase show increasing spe-cific activities toward the end of ripening and overripening. The third group (C) iscomposed of only β-D-xylosidase, whose activity remains constant from the pre-ripeuntil the overripe stage. A large increase in β-D-galactosidase, α-L-arabinopyranosi-dase and a limited increase in β-D-glucosidase occur during later stages of ripening,which could degrade the arabinogalactan side chains on the pectic fraction of thefruit cell walls (Fils-Lycaon and Buret, 1991). Watermelon fruit are rich in α-mannosidase and β-N-acetyl-hexosaminidase, and changes in their cell walls appearto be less pronounced than in muskmelons (Nakagawa et al., 1988).

PG isozymes play an important role in postharvest fruit decay caused by micro-bial pathogens. Eight PG isozymes, with pIs ranging from 3.7–8.6, were isolated inPhomopsis cucurbitae infected fruit (Zhang et al., 1997). The most prominent isozymehas endo-PG activity, a molecular weight of 54 kDa, a pI of 4.2 and an optimumactivity pH of 5 (Zhang et al., 1999a). One of 14 PG isozymes potentially producedby Fusarium solani was detected in infected muskmelon tissue (Zhang et al., 1999b).The endo-PG isozyme has a molecular weight of 38 kDa and pI of 9.5.

Pear

Endo- and exo-PG are present in pear (Pressey and Avants, 1976). A rise in endo-PG activity during ripening correlates with the decrease in polymerization of pectinin the fruit and release of uronic acid in the fruit (Pressey and Avants, 1976; Bartleyet al., 1982). Yoshioka et al. (1992) found that de-esterification of polyuronides witha high degree of methoxylation is mainly responsible for the solubilization ofpolyuronides in softening of pears. The softening characteristics of pear, however,vary with cultivar. A comparison of enzymatic activities during the softening ofChinese pear cv. ‘Yali’ (Pyrus bretschneideri) fruits, Japanese pear cv. ‘Nijisseiki’(P. pyrifolia) and European pears ‘La France’ and ‘Bartlett’ (P. communis) by Ninget al., (1997) demonstrates differences that are cultivar specific. Rapid increases ofPE and PG activities and water-soluble pectins occurred with a decrease in HCl-soluble pectins in ‘La France’ and ‘Bartlett’ fruits, whereas enzyme concentrationsand an increase in water-soluble pectins did not occur to the same extent in ‘Yali’and ‘Nijisseiki’ fruits. The latter cultivars also had slight decreases in HCl-solublepectin. Cellulase activity increased in ‘La France’ and ‘Yali’ fruits, whereas itsincrease was slight during ripening in ‘Nijisseiki’ and ‘Bartlett’ fruits. Ahmed andLabavitch (1980) did not find Cx activity in ripening Bartlett pears. Yamaki andMatsuda (1977) reported endo-PG isozymes with pH optima of 5.5 and 7.0 in orientalpears (P. serotina). The neutral PG isozyme increased with ripening and so didβ-glucosidase.

Two PG isozymes are present in ripening ‘D’Anjou’ (Pyrus communis L.) pears.One of these hydrolyzes the pectate chain randomly with a pH optimum of 4.5,while the major PG catalyzes a stepwise removal of monomer units from the

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nonreducing ends of the substrate molecules. This endo-PG that exhibits high affinityfor pectate has a pH optimum of 5.5 and is activated by Ca2+ and Sr2+ (Pressey andAvants, 1976). Three isoforms of β-galactosidase also appear to have distinct regu-latory mechanisms during ripening and softening of ‘D’Anjou’ pear. β-Galactosidasemeasured in cell walls at various stages of ripening at 20°C exhibits three distinctregions of cell wall β-galactosidase activity between pH 8.6–8.7, pH 7.1–7.7 andpH 5.6–5.7. The activity of the basic pI group declines slowly, while the neutral pIgroup declines sharply after peaking at four days. The activity of the acidic pI groupthat is initially low, steadily increases with ripening (Perdue et al., 1998).

Two general types of polyuronides, the major type being a homogalacturonan(HGA) whose molecular weight decreases upon ripening, are found in ‘Bartlett’pears. The other type comprises heteropolymers composed of various amounts ofarabinose, rhamnose and galactose. Glycosyl-linkage analysis of the arabinosyl-polyuronide gave results indicating a rhamnogalacturonan I-like polysaccharide withlarge, highly branched araban side chains (RG-I). There does not appear to be alinkage between HGA and RG-I, but highly branched araban RG-I in ripening pearsappears to be degraded with the initial loss of much of its arabinose side chains(Dick and Labavitch, 1989). PG, α-galactosidase and α-mannosidase activities alsoincrease with softening of ‘Bartlett’ pears (Ahmed and Labavitch, 1980).

Peach

Peach softening seems to result mainly from PG activity. Clingstone varieties thatremain firm when ripe exhibit only exo-PG, while freestones that develop a softmesocarp during ripening show increased exo-PG and endo-PG activity (Downs andBrady, 1990), as well as cellulase (Cx) activity (Hinton and Pressey, 1974). Thecorrelation suggests that the extensive softening of the mesocarp tissue of freestonepeaches is related to the presence of endo-PG activity (Pressey and Avants, 1978).Cx, exo- and endo-PG activities are very low at the preclimacteric stage of freestonepeaches, and significant increases occur only after the ethylene rise. During fruitdevelopment, cellulase and exo-PG activity are high at the first stage of fast growth,while endo-PG activity increases during the last growth stage when the fruit hasreached its final size and ripening has started (Zanchin et al., 1994; Bonghi et al.,1998; Ruzzon et al., 1998). Endo-β-1,4 glucanase (Egase) appears to play a primaryrole during early growth and at the beginning of softening, while PG may be involvedduring melting (Bonghi et al., 1998; Ruzzon et al., 1998). Propylene treatmentsreduce EGase activity during the early stage of fruit growth but then dramaticallyenhance this enzyme activity with the onset of ripeness and ultimately accelerateloss of firmness (Bonghi et al., 1998). This observation and the presence of twoisoforms (pI 6.5 and 9.5) suggest that different EGase genes operate during the earlyand late developmental stages in peach fruit. The isoform with the higher pI pre-dominates during late development. α-1,4-Galacturonase (EC 3.2.1.15) is alsoinvolved in softening of “melting flesh” peaches during the latter stages of ripening,causing the destruction of cell wall polymers containing long, thin pectin aggregates,while leaving cell wall polymers containing short, thick pectin aggregates intact(Fishman et al., 1993). Orr and Brady (1993) reported that the pattern of accumulation

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of endo-PD activity in peach differed from that observed in tomato, but like intomato, endo-PG is not the sole determinant for textural changes. A small level ofactivity was detected in fruit that was substantially softer than mature unripe fruitin which little activity was detected. In very soft fruits with the “melting soft”character, however, enzyme activity increased sharply.

Cellulose activity in preclimacteric fruits is attributed to one molecular form (pI6.5). Two main forms are present at the postclimacteric stage (pI 6.5 and 9.5, thelatter being the most abundant). Endo-PG is present at the preclimacteric stage intwo molecular forms (pI 5.2 and 8.4). At the postclimacteric stage, the acidic formdisappears, and activity is due exclusively to the pI 8.4 form. Exo-PG activity at thepreclimacteric stage is present as one form (pI 4.9). A reduction in the activity ofthe acidic form occurs at the postclimacteric stage concurrently with the appearanceof another isoform (pI 8.8) (Tonutti et al., 1994). The more abundant form of theenzyme is a polypeptide with a molecular weight of 66 kDa and with a substantialexcess of basic over-acidic amino acids (Downs et al., 1992).

Storage temperature influences pectic enzyme activity and peach fruit firmness.Firmness decreases more rapidly at lower temperatures. Initial storage at 1°C fol-lowed by 20°C also causes a decrease in PE and an increase in PG activity duringstorage and ripening (Salmeron and Artes, 1991). The poor texture described as“woolliness” is apparently caused by altered pectic polymer breakdown by inhibitedPG activity that does not affect PE activity. Cold-stored fruits show lower PG activity,which likely causes the lowering of lower water-soluble pectin content and increasessodium carbonate soluble pectin content. PE activity does not appear to be affectedby cold storage (Choi and Lee, 1999; Sonego et al., 1998). The onset of woollybreakdown could be delayed, and its severity could be reduced by exposure toethylene at 1 or 10 µl/L, and this does not affect fruit softening. Sonego et al. (1998)reported undetectable PG activity in control fruits during storage at 0°C, but PGactivity was induced by ethylene at 0°C and enhanced five- to 10-fold after transferto 20°C. However, PG protein content was unaffected by ethylene treatment. PEcontent and activity, which increased during cold storage, were also unaffected byethylene treatment. ‘Yumyeong’ peach fruits stored at 0°C, with or without a mod-ified atmosphere (MA, using polyethylene film, 0.03 mm thickness), for four weeksfollowed by a ripening period at 20°C indicated a lack of relationship between PEactivity and EDTA-soluble pectin content with wooliness. Reduction in woollinessoccurred with MA storage and appeared to be based on an increase in water-solublepectin and a decrease in sodium-carbonate-soluble pectin by increased PG activity(Choi et al., 1997).

Chang et al. (1999) recently demonstrated the involvement of an array of enzymesin the rate of softening of peach fruits using a fast-softening cultivar (‘Mibaekdo’)and a slow-softening cultivar (‘Yumyung’). They found activities of soluble and cell-wall-bound PG to be similar in both cultivars. Activities of soluble and cell-wall-boundα-galactosidase, β-galactosidase, β-arabinosidase and α-mannosidase were main-tained at high levels in both cultivars, while those of β-glucosidase and β-xylosidasewere low during softening. However, higher activities of soluble α-galactosidase, β-arabinosidase and α-mannosidase were observed in ‘Yumyung’ cultivar relative to‘Mibaekdo.’ ‘Yumyung’ fruit exhibited lower cell-wall-bound α-galactosidase and

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β-arabinosidase activities than ‘Mibaekdo’ throughout softening. The cell-wall-bound α-mannosidase activity was similar in both cultivars, but cell-wall-bound β-galactosidase was higher in ‘Mibaekdo’ cultivar than in ‘Yumyung’ cultivar.

Tomato

Most of the reported work on ripening and softening of fruits has been on tomato,because the fruit provides the most obvious change in color from green to red, ripensuniformly in a manner that could easily and consistently be staged using morpho-logical markers and is a rich source of pectic enzymes. The softening of tomato hasbeen well reviewed (Gray et al., 1992; Gross, 1990; Giovannoni et al., 1992; Krameret al., 1989; Pressey, 1986). The richest plant source of PG is ripe tomato fruit.Preclimacteric tomatoes have very low levels of exo-PG and no detectable endo-PG(Pressey, 1987). As fruit begins to change color, PG activity increases. Endo-PG appearsat the onset of ripening and increases sharply during ripening. In ripe tomatoes, thelevel of endo-PG is about 600 times higher than that of exo-PG in green fruit (Pressey,1986) and becomes a major protein in the ripe fruit (Brady et al., 1982). PG accumu-lation parallels pectin degradation and fruit softening. Consequently, endo-PG has beenimplicated as a primary determinant of pectin degradation and softening of tomatofruit (Huber, 1983; Grierson, 1985; Brady et al., 1987; Giovannoni et al., 1989). PGactivity is highest in the outer locule wall of the pericarp tissue followed by theinner locule walls and the placental tissue. The enzyme is not present in the locularcontents. Activity first appears in the placenta and then develops in both the innerand outer locule walls as the change in color spreads to the pericarp (Pressey, 1977).The deep red color in tomatoes has been associated with high PG levels. The inabilityof accumulated PG early in ripening to depolymerize pectin appears to result fromthe fact that PG deposition during early ripening is uniform and mobile (Brady et al.,1987). There is less correlation between other cell wall degrading enzymes and therate of tomato fruit softening (Tigchelaar et al., 1978; Wallner and Walker, 1975).PE for example, increases severalfold during ripening, and several isozymes exist,with one being the dominant isozyme (Pressey and Woods, 1992), but there is nosignificant correlation between firmness and pectinase in tomato. A number of otherfactors contribute to softening in tomatoes, and there is considerable evidence thatPG alone cannot be used as a determinant of softening. Cellulotic enzymes degradecellulose and hemicellulose, and the amounts of these enzymes have been correlatedwith softening (Pressey, 1977; Huber, 1983). Endo-PG-dependent pectin hydrolysismay generate oligosaccharide molecules capable of influencing other aspects of thesoftening process (Brady et al., 1987; Bennett and DellaPenna, 1987; Baldwin andPressey, 1988). This includes the production of ethylene by cell wall fragments(Baldwin and Pressey, 1990). In very ripe fruit, pectin depolymerization could resultfrom leakage of calcium chelating compounds such as citrate and malate into avail-able free space. PG action in vitro is severely limited by the presence of calcium inthe wall (Brady et al., 1987). Using a pleiotropic genetic mutation, rin, that blocksmany aspects of ripening including PG synthesis, and inserting another PG geneunder an inducible promoter, Giovannoni et al. (1989) demonstrated that accumu-lated PG had no significant effect on fruit softening, ethylene evolution or color

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development, in spite of its role as the primary determinant in cell wall pectindegradation. This observation might, however, be the result of the pleiotropic effectsof the rin that inhibit other normal PG-induced changes as they occur in the normalfruit (Langley et al., 1994; Fenwick et al., 1996).

Three main PG isozymes with different physical properties have been identifiedin tomato (Ali and Brady, 1982). The isozymes have in common only one polypep-tide, but differ in glycosylation and composition of the inactive units (Moshrefi andLuh, 1983; Pressey, 1984). During early ripening, a PG isoform (PG1) of approxi-mately 110 kDa develops. As fruit development continues, two smaller isoforms(PG2a and PG2b) of approximately 42 and 46 kDa, respectively, accumulate (Bradyet al., 1982). Both have the same isoelectric point (9.4), whereas PG1 has a pI of8.6. PG1 is also more thermostable than PG2 (Knegt et al., 1988; Pogson and Brady,1993). The relative amounts of PG isozymes vary widely. PG1 is thought to be acomplex of PG2a or PG2b with a further polypeptide known as the β-chain and canbe broken down to yield PG2 and the β-subunit in a 1:1 ratio (Hobson and Grierson,1993). It has been suggested that the softening occurs from the combination of PG2with the β-form to form PG1. PG1 content appears to be better correlated with themaceration process (Brady et al., 1985; Pogson and Brady, 1993). A nonspecificconverter glycoprotein, capable of converting PG2a and PG2b into PG1, that islocalized in the plant cell wall has been purified (Pressey, 1994). Alkaline hydrolysisof PG1 results in the release of PG2. This suggests that the converter is the associatedmolecule (Pressey, 1986). The purified converter can also be reacted with PG2enzymes to form an enzyme similar to PG1 (Knegt et al., 1988). Thus, tomatosoftening appears to be related to the interactions between PG2 polypeptide and theimmobile converter peptide to form the physiologically active PG1 enzyme. Chilling-associated softening, however, is more related to PE activity. Marangoni et al. (1995)demonstrated that while softening of nonchilled fruit was well correlated withextracted PG1 activity, chilling-associated softening correlated with higher initialextracted PE activity. The loss of turgor from translocation of water to the PE-modified cell wall was suggested to be responsible for softening as a result ofchilling.

Several reports correlate PG activity with shelf life and survivability of the fruit.Low PG levels increase the average molecular weight of pectin and improve firmnessthroughout ripening (Murray et al., 1993; Young, 1995; Poole, 1993). They alsorender the fruit much less susceptible to mechanical damage and cracking (Schuchet al., 1991). Fruits with reduced PG also have increased resistance to Geotrichumcandidum and Rhizopus stolonifer, fungi that normally infect ripening fruits (Krameret al., 1992).

CONCLUSION

Biochemical and physiological consequences of fresh-cut processing are related tothe wounding of tissue that occurs. The destruction of surface cells and injury stressof underlying tissues cause reactions that result in sensory deteriorations such asoff-flavor, discoloration and loss of firmness. Many factors potentially influence thenature and extent of enzymatic activities and their effect on the flavor and texture

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Enzymatic Effects on Flavor and Texture of Fresh-cut Fruits and Vegetables 163

of fresh-cut fruit and vegetable products. These include growing conditions andcultural practices, cultivar and maturity at harvest, harvesting and handling methodsand storage conditions. It is evident from this review that the continued growth ofthe fresh-cut industry will demand a better understanding of enzymatic changes andhow they affect sensory and shelf life properties of processed products. It is alsoimportant that interactions of the enzymes with other food components be understoodso that beneficial properties could be optimized and detrimental effects could beminimized. Most of the reported properties and characteristics of enzymes in com-mon fresh-cut processed fruits and vegetables are from the uncut produce. Whilethese serve as a valuable pool of information for the fresh-cut industry, continuedindustry-specific research will ensure improved sensory quality and shelf life andconsistency of fresh-cut fruit and vegetable products.

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Microbiology of Fresh-cut Produce

Gillian M. Heard

CONTENTS

IntroductionGeneral Microbiology of Fresh-cut Produce

Total MicrofloraOrigins of the Microflora of Fresh-cut Produce

Contamination on the FarmPreharvestPostharvest

Contamination During ProcessingFactors Affecting the Growth of Microorganisms

Handling Practices on the FarmWater Quality DisinfectantsFertilizersDamageRainfall and Temperature

Conditions During Processing/PackagingProcesses SanitationTemperaturepHPackaging

Conditions During RetailingOther Factors

Microbial Spoilage of Fresh-cut Products Effect of Microbial Growth on the Quality and Shelf Life of Fresh-cut SaladsSpoilage Characteristics Occurrence of Spoilage Organisms in Fresh-cut ProductsCharacteristics of Spoilage Organisms

Pseudomonads and Related SpeciesLactic Acid BacteriaEnterobacteriaceae

7

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Coryneform BacteriaArthrobacter

Yeasts and Molds Foodborne Pathogens

The Association of Foodborne Pathogens with Fresh-cut SaladsPathogens of Concern

Listeria Monocytogenes

Enteric Pathogens (Family Enterobacteriaceae)

Escherichia Coli

Shigella

Salmonella

Spore-Forming Bacteria

Staphylococcus Aureus

Campylobacter

Yersinia Enterocolitica

Aeromonas

Species

Vibrio

SpeciesVirusesParasites

Detection of Pathogens in Fresh-cut ProductsBiocontrol

BiopreservationUse of Natural Antimicrobial Compounds

BacteriocinsNatural Plant Volatiles

Induced ResistanceSynthesis of Phenolic Secondary Metabolites—Ligninand Phytoalexins Synthesis of Pathogenesis-Related ProteinsInduced Resistance

ConclusionReferences

INTRODUCTION

Microorganisms are natural contaminants of fresh produce and minimally processedfresh-cut products, and contamination arises from a number of sources, includingthe farm environment, postharvest handling and processing (Beuchat, 1996; Heard,1999b). Fresh-cut products are particularly susceptible to microbial attack becauseof the changes occurring to the tissues during processing. Processing operationssuch as cutting, shredding and slicing not only provide opportunities for contami-nation but also cause damage to fruit and vegetable tissues and cellular structure,leading to leakage of nutrients and cellular fluids. Unlike other types of processing,such as freezing or canning, no heat treatment is given to the produce to reducemicrobial populations. Fresh-cut products are also packaged under modified atmosphere

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Microbiology of Fresh-cut Produce

189

conditions and stored refrigerated for up to 10–15 days. This creates a favorableenvironment and time for proliferation of spoilage organisms and microorganismsof public health significance (Ahvenainen, 1996; Francis et al., 1999). Microorgan-isms impact the economic value of fresh-cut products by decreasing product shelflife, through spoilage, and by posing a risk to public health by causing foodbornedisease (Doyle, 1990; Lund, 1992; Brackett, 1994; Nguyen-The and Carlin, 1994,2000). It has been known for more than a century that raw fruits and vegetables canact as vehicles for outbreaks of human disease. However, until recently, there has beenlittle interest in documenting evidence of the incidence of foodborne pathogens onfresh produce. Additionally, outbreaks of foodborne disease associated with fruitsand vegetables have rarely been documented (Beuchat, 1998). Subsequently, the eco-logical development and activity of microorganisms on produce and, in particular,on fresh-cut products is still poorly understood. Current knowledge is mostly limitedto qualitative descriptions of the microbial species isolated at the time of spoilageor after an outbreak of foodborne disease (Tauxe et al., 1997; Heard, 1999b). Alsolacking is our understanding of the factors affecting microbial contamination andcolonization of fresh produce and processed fresh cuts, microbial interactions onthese products and biochemical changes occurring. What do the microorganismsgrow on and what metabolites are produced and do these metabolites contribute tothe spoilage process? Does the growth of spoilage bacteria such as pseudomonadsand lactic acid bacteria influence the growth of pathogenic species? Are there naturalbiocontrol mechanisms that can assist in controlling the microflora? This informa-tion, as well as knowledge of the factors affecting the growth of microorganisms onminimally processed fruits and vegetables, is necessary if we are to control thequality and safety of fresh-cut products.

The aims of this chapter are to review the current available knowledge on the diversityof the microflora of fresh-cut products, the source and significance of the predominantmicrobial species, the conditions that encourage microbial growth and the consequencesof microbial growth. Plus, recommendations for future research will be made.

GENERAL MICROBIOLOGY OF FRESH-CUT PRODUCE

T

OTAL

M

ICROFLORA

Fruits and vegetables become contaminated with microorganisms while on the plant,in the field, during harvest and transport to market and during processing and pack-aging. Microorganisms may be present as chance contaminants, or they may possesscharacteristics enabling colonization of the plant. They may cause spoilage or may beof public health significance. Hence, the microflora associated with fresh-cut produce isdiverse. Much of the literature reporting the occurrence of microorganisms in theseproducts has, unfortunately, only focused on total bacterial populations and microbialgroups, such as coliforms, fecal coliforms, pectinolytic species and yeast and moldcounts. Although we know which pathogens may occur in fresh-cut products, ratherthan monitoring pathogen populations directly, coliforms are often used as indicatorsof contamination with pathogens from fecal sources. Unfortunately, many nonpathogenic

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bacterial species normally present on the surface of fresh produce, such as species of

Enterobacter

, will give positive results on coliform detection. It is now increasinglyacknowledged that coliform populations of raw and freshly processed vegetablesshould not be used to indicate contamination with fecal pathogens (Beuchat, 1998;Nguyen-The and Carlin, 2000). Nevertheless, total count and coliform tests are simpleto perform, and they are used by the fresh-cut produce industry as indicators of hygieneand quality. For this reason, such data have been summarized for a wide range offresh-cut products and are shown in Table 7.1. Numbers of mesophilic organismsreported on fresh-cut salad vegetables at the time of processing are similar to thenumbers present on unprocessed produce. Microbial counts are within the range10

1

–10

9

cfu/g, varying with fruit and vegetable type (Table 7.1). Approximately 80–90%of these organisms are reported to be gram-negative rods, which are predominantlypseudomonads. Approximately 10–60% of these organisms are fluorescent, pectinolyticpseudomonads, varying from 10–20% of isolates from shredded lettuce to 20–60% offluorescent pseudomonads isolated from carrots and endives (Nguyen-The and Carlin,1994, 2000; Carlin et al., 1989; Nguyen-The and Prunier, 1989; Magnusson et al., 1990;Bennick et al., 1998; Jayasekara, 1999). Fluorescent pseudomonads are the dominantgroup isolated from endive (Jacques and Morris, 1995), spinach, cauliflower and carrots(Garg et al., 1990).

A recent study of the microflora of chicory and mung bean sprouts (Bennick et al.,1998) also reported predominance by pseudomonads. Enterobacteriaceae are alsopresent on minimally processed vegetables, however, their presence is often summarizedas coliform or fecal coliform counts (Nguyen-The and Carlin, 1994; Bennick et al.,1998). Other microbial groups reported on fresh-cut vegetables include yeast and molds(Table 7.1). For example, molds are reported at populations varying from 10

2

cfu/g oncut lettuce to 10

8

cfu/g in shredded vegetable packs and shredded carrots. In mayonnaise-based salads, yeast populations as high as 10

6

have been reported, the low pH favoringyeast growth over other microorganisms (Christiansen and King, 1971; Fowler andClark, 1975; Brocklehurst et al., 1983; Brocklehurst and Lund, 1984; Lindroth et al.,1985; Hunter et al., 1994). Lactic acid bacteria are present on selected fresh-cut vege-tables and have been reported to occur in populations ranging from 10

2

cfu/g in shreddedchicory to 10

6

cfu/g in mixed vegetables (Manzano et al., 1995; Jacxsens et al., 1999). There are few reports of the predominant microflora of fresh-cut fruit products.

Unlike on whole fruits, growth of molds on fresh-cut products does not appear tobe a major problem, and it may be assumed that the high moisture content of ready-to-eat fruit products may encourage faster-growing bacteria and yeast. Yeast popu-lations of 10

3

–10

4

have been reported for processed fruits (Nguyen-The and Carlin,1994), and total bacterial populations of 10

6

cfu/g have been reported for cantaloupe(Sapers and Simmons, 1998). More detailed investigations of the occurrence ofmicroorganisms in processed fruit products are needed.

O

RIGINS

OF

THE

M

ICROFLORA

OF

F

RESH

-

CUT

P

RODUCE

Microbial contamination of fruits and vegetables is reported to arise during growth;from the soil, organic matter, organic fertilizers, irrigation processes, insects, animalsand human contact; and from postharvest practices, including washing, trimming

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TABLE 7.1Total Microbial Populations and Predominant Microbial Groups Present on Fresh-cut Fruits and Vegetables

1

Microbial Populations (Log cfu g−−−−

1

)

Fresh-cut Product

Total Count (Mesophilic)

Coliform Count

Lactic Acid Bacteria

Yeast and Molds Ref.

Individual Commodities

Broccoli florets 6.5 5.9

2

— 5.2 Brackett (1989)Broccoli florets 4.7 2.2 — 3.3 Mohd-Som et al.

(1994)Broccoli florets 4.7 2.1 — 3.25 Jacques and Morris

(1995)Cantaloupe 6.11 — — — Sapers and Simmons

(1998)Minimally processed cantaloupe

1.05 — ND — Lamikanra et al. (2000)

Cut carrots 4.4 — — — Priepke et al. (1976)Carrot sticks 4.99–5.77 — ND–3.1 ND–4.25 Garg et al. (1990)Shredded carrots 2.9 — 1.1 — Chervin and Boisseau

(1994)Shredded carrots 6.00 — 4.00 <2.00 Carlin et al. (1990)Carrot sticks 4.98 2.84 ND — Garg et al. (1993)Shredded carrots 4.88 — 3.52 3.15 Kakiomenou et al.

(1996)Carrot sticks 5.13 — — — Odumeru et al. (1997)Shredded carrots 2.84–3.85 1.54–2.3

(fecal)1.65–2.69 2.00–2.12 Sinigaglia et al. (1999)

Minimally processed broad-leaf endive

3.83–4.82 — — — Carlin et al. (1996)

Cut chicory endive 4.00 — — — Bennick et al. (1998)Chicory endive (shredded)

5.2 — 2.63 3.0 Jacxsens et al. (1999)

Coleslaw <4.00–>8.00 <2.00–>6.00 — — Fowler and Foster (1976)

Cabbage (coleslaw)

4.07–7.08 — ND–2.4 ND–2.2 Garg et al. (1990)

Coleslaw mix 5.14 — — — Odumeru et al. (1997)Coleslaw (dryslaw)

7.32–7.84 — — — Jayasekara (1999)

Cut celery 5.7 — — — Priepke et al. (1976)Cut lettuce 5.3 — — — Priepke et al. (1976)Prepared lettuce for caterers

5.6 3.9 Maxcy (1978)

Lettuce 5.41 — ND ND Garg et al. (1990)

(

continued

)

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TABLE 7.1Total Microbial Populations and Predominant Microbial Groups Present on Fresh-cut Fruits and Vegetables

1

(Continued)

Microbial Populations (Log cfu g−−−−

1

)

Fresh-cut Product

Total Count (Mesophilic)

Coliform Count

Lactic Acid Bacteria

Yeast and Molds Ref.

Lettuce 6.39–7.69 4.14–5.29 — — Gras et al. (1994)Chopped lettuce 4.85 — — — Odumeru et al. (1997)Processed lettuce 2.5–6.2 — — — Francis and O’Beirne

(1998)Shredded lettuce 4.28 — <1 2.07 Delaquis et al. (1999)Lettuce salad 7.23–7.61 — — — Jayasekara (1999)Chopped bell peppers

3.5 — — — Izumi (1999)

Fresh-cut mushrooms

8.3 — — — Sapers and Simmons (1998)

Potato strips 2.00 — — — Gunes et al. (1997)Sliced potatoes 2.01–2.6 <0.7 <0.7 — Laurila et al. (1998)Diced potatoes 5.00 — — — Izumi (1999)Potato salad 5.41–4.98 — — — Jayasekara (1999)Japanese radish shreds

3.9 — — — Izumi (1999)

Trimmed spinach leaves

4.00 — — — Izumi (1999)

Mixed Products

Chicken and vegetable salad

5.8 3.27 — 4.00 Christiansen and King (1971)

Green salad <4.00–7.00 <2.00–>6.00 — — Fowler and Foster (1976)

Ham and vegetable salad

6.17 2.47 — 3.08 Christiansen and King (1971)

Mixed green salad <4.00–8.00 <2.00–6.00 — — Fowler and Foster (1976)

Mixed vegetable salad

5.00 4.00 3.00 — Manvell and Ackland (1986)

Mixed vegetables 8 — 5.5–6.3 4–4.2 Manzano et al. (1995)Mixed salad in school kitchens

1.84–2.99 0.7–1.90 — — Martínez-Tomé et al. (2000)

Packaged garden salad (iceberg lettuce, carrot, red cabbage)

5.3–8.9 — — 0.9–3.85 yeasts

<0.3–2.2 molds

Hagenmaier and Baker (1998)

Prepackaged ready-to-serve salad

5.5–8.3 <3–6.75 Lack et al. (1996)

Raw vegetables 5.7 2.3 — — Kaneko et al. (1999)

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and packing (Beuchat, 1996). Fresh-cut produce may be further contaminated duringtransport, from processing steps and during packing (Heard, 1999b). This sectiondiscusses the source of the microflora and highlights the gaps in our knowledgeabout where the contamination actually occurs along the production chain.

Contamination on the Farm

Preharvest

Fruits and vegetables become contaminated in the field during growth. The micro-flora may arise from within the plant, such as from the seed or tuber or from theenvironment, during growth. Seeds are a source of foodborne pathogens such as

Bacillus cereus

and

Salmonella

as well as bacteria and fungi that cause postharvestdiseases (Portnoy et al., 1976; Maud, 1983; Harmon et al., 1987; O’Mahony et al.,1990; Nguyen-The and Carlin, 2000). Thus, the first leaves emerging from contam-inated seeds will also be contaminated. For example, Morris and Lucotte (1993)reported total populations of 10

3

cfu/cm

2

on the first leaves of a green endive plant.Most contamination occurs on the outside or surface of plants, although in somefruits and vegetables, the inner tissues may be invaded in the early stages of fruitdevelopment (ICMSF, 1998). The predominant microbial species present on fresh-cut produce (Table 7.1) are also present in soil, irrigation water and the general farmenvironment. Table 7.2 summarizes the sources of microbial contamination duringthe production of fresh-cut produce, as reported in the literature. As mentionedpreviously, pectinolytic and fluorescent bacteria, particularly the pseudomonads, arethe dominant flora of many plant products at the time of harvest. These organismsas well as coryneform bacteria, lactic acid bacteria, yeasts and molds are derivedfrom air, water and soil and contaminate the leaves and outer surfaces of plantsduring growth (Lund, 1983, 1992; ICMSF, 1998).

Crops can become contaminated with spoilage organisms from a number ofsources on the farm. The presence of rotting organic material in soil should be

TABLE 7.1Total Microbial Populations and Predominant Microbial Groups Present on Fresh-cut Fruits and Vegetables

1

(Continued)

Microbial Populations (Log cfu g −−−−

1

)

Fresh-cut Product

Total Count (Mesophilic)

Coliform Count

Lactic Acid Bacteria

Yeast and Molds Ref.

Ready-to-use mixed salad

7.18 6.60 5.3 — Vescovo et al. (1995)

Salad mix 5.35 — — — Odumeru et al. (1997)Vegetable salad 4–7 — — — Wright et al. (1976)

1

Products were sampled prior to storage.

2

Reported as Enterobacteriaceae counts.

—Not sampled.

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TABLE 7.2Sources of Microbial Contamination of Fresh-cut Salad Products—from the Farm and during Processing

Source of Contamination

Product Example Microflora Ref.On the Farm—Pre- and Postharvest

Grazing domestic animals, manure from cattle (unprocessed organic fertilizer)

Vegetables Spoilage flora and pathogens—

Salmonella,

E. coli

O157:H7,

Cryptosporidium parvum

Tauxe et al. (1997), Nguyen-The and Carlin (2000)

Hygienic practices of farm workers

Sliced melon

Vibrio cholerae

Ackers et al. (1997)

Raspberries and sliced melons

Pathogens Ackers et al. (1997), Lund and Snowden (2000)

Insects Damaged fruits and vegetables

Spoilage or pathogenic microflora

Lund (1983)

Overhead irrigation systems

Tomatoes Bacterial contamination Samish and Etinger-Tulczynska (1963)

Packing shed design All produce — Troller (1983), Coghlan (2000)

Pesticides (mixed up using contaminated water)

Fresh fruits and vegetables, e.g., raspberries (inoculated with pathogens)

Salmonella

,

E. coli

O157:H7,

Shigella

survival and growth in the pesticide,

Cyclospora cayetanensis

on raspberries

Hertwaldt et al. (1997), Tauxe et al. (1997), Coghlan (2000)

Rainfall and temperature

Vegetables Lactic acid bacteria numbers are low in hot, dry conditions

Mundt et al. (1967), Mundt (1970)

Rhizospheres Root crops Fungal spores, Pectolytic pseudomonads, spoilage microflora

Droby et al. (1984), Sands and Hankin (1975)

—Seeds, tubers Endive plants Total microflora Morris and Lucotte

(1983)Sprouts

Salmonella,

Bacillus cereus

Portnoy et al. (1976), Maud (1983), Harmon et al. (1987), O’Mahony et al. (1990)

Soil, organic material Rhizospheres

Cl. perfringens

Lund (1992)Transport—trucks, pallets, boxes, crates

— — No reports

*

Wastewater, sewage Vegetables

Vibrio cholerae,Listeria monocytogenes

Beuchat (1998), Nguyen-The and Carlin (2000)

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avoided, as it may provide a source of contamination for nearby growing crops. Forexample, pectolytic pseudomonads have been shown to survive in soil containingthe rhizospheres of previous crops (Sands and Hankin, 1975). This may result inspoilage of subsequent crops (Nguyen-The and Carlin, 2000). In a review by Lund(1992), it was suggested that rhizospheres may also support the survival of clostridiaup to 10

6

cfu/g.

Clostridium

perfringens

has been reported as a common species inrhizospheres. Harvesting equipment can become contaminated with fungal sporesand bacteria, from the soil and from decaying organic matter, subsequently, contam-inating new crops. For example, Droby et al. (1984) reported that potato tubersbecame contaminated with fungal spores originating from infected foliage.

Domestic animals may also disseminate spoilage organisms ingested with plantfodder (Nguyen-The and Carlin, 2000). Insect activity is encouraged by the presenceof decaying organic matter remaining in fields, and the insects may disseminate

TABLE 7.2Sources of Microbial Contamination of Fresh-cut Salad Products—from the Farm and during Processing (Continued)

Source of Contamination Product Example Microflora Ref.

Cucumbers Fecal coliformsWind, dust — — No reports*

During Processing

Equipment—trimming, shredding, cutting, surfaces

Vegetables Total microflora and coliforms

Garg et al. (1990), Barry-Ryan and O’Beirne (1998)

Hygiene of handlers — — No reports*Non-vegetable ingredients

Chicken or meat added to salads

Total microflora increase Christiansen and King (1971)

Packaging Lettuce Wider variety of microorganisms present in unsealed packages than in sealed packages

Magnuson et al. (1990), King et al. (1991)

Use of chlorine All produce Total counts reduced by 1–3 log

Garg et al. (1990), Nguyen-The and Carlin (1994)

Broccoli Delayed spoilage by pseudomonads in modified atmosphere packages

Brackett (1989)

Wash water, water bath used by the packer

Tomatoes Total microflora increase,

Salmonella

(caused an outbreak of foodborne disease)

Tamplin (1997)

* There is little evidence to document the effect of these factors as sources of contamination, but theyare commonly referred to in reviews by Beuchat (1996, 1998), ICMSF (1998) and Heard (1999b).

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microorganisms to other crops, resulting in contamination (Lund, 1983). The use ofoverhead irrigation systems has been linked with bacterial contamination of fruitsuch as tomatoes. Bacteria enter the fruit through the sepals (Samish and Etinger-Tulczynska, 1963). The role played by the wind for contaminating fruits and vege-tables is not reported in the literature and warrants investigation. Wind may transferdust contaminated with mold or bacterial spores on the surface of plants.

Pathogens of public health significance, including

Listeria monocytogenes

, ther-motolerant

Campylobacter

and the opportunistic pathogen

Pseudomonas aeruginosa

,have also been isolated from soil or water, bird and animal droppings (Geldreich andBordner, 1971; Colburn et al., 1990; Park and Sander, 1992; Nguyen-The and Carlin,2000) and farm workers in the fields. Farmers and agricultural laborers often assumethat because raw produce is soiled during growth, personal hygiene and equipmentcleanliness is not necessary. Inadequate hand washing, disposal of domestic waste andinadequate cleaning of farm equipment can result in contamination of produce withspoilage organisms and possibly with microorganisms of public health significance(Geldreich and Bordner, 1971; Beuchat, 1996, 1998; Brackett, 1999). The

Guide toMinimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables

(Guidancefor Industry, 1998) has recently been published by the U.S. Department of Agricultureto assist in educating farmers and processors in producing safe produce. These guide-lines address the hygiene issues mentioned above.

Wastewater or water polluted with fecal material is also a source of contamina-tion. Similarly, the use of untreated organic fertilizers and direct application of humanfecal material to growing crops may result in contamination with pathogens (ICMSF,1998). Pathogens, including members of the Enterobacteriaciae, viruses, protozoaand nematodes and

L. monocytogenes

may be transmitted to fresh fruits and vege-tables from sewage water and untreated wastewater and fecal matter. For example,wastewater-irrigated vegetables are reported to be responsible for cholera outbreaksin Chile and Costa Rica in the early 1990s (Nguyen-The and Carlin, 2000). Severalreports of pathogen contamination of fruits document the source of the pathogens,

Salmonella, Escherichia coli

O157:H7 and

Cryptosporidium parvum

, as manurefrom grazing cattle (Tauxe et al., 1997). Several investigations of the influence ofwastewater on microbial populations of irrigated vegetables have been reported inthe literature, although there are no recent studies. Sadovski et al. (1978), duringan evaluation of methods for irrigating crops, recorded fecal coliform counts of10

3

cfu/100 g on cucumbers taken from sewage-irrigated plots. Rosas et al. (1984)investigated the bacteriological quality of crops irrigated with wastewater in an areaof Mexico City.

Postharvest

Fruits and vegetables can become further contaminated during harvest and frompostharvest handling from the handlers, the work surfaces, wash water, packagingcrates and pallets and trucks during transport (Table 7.2). Improper hygiene practicesmay influence the microbial safety of produce during harvest (Geldreich and Bordner,1971). Toilets should provide adequate hand washing facilities, and sewage shouldnot be in contact with crops (Beuchat, 1998). Contamination of fruits such as rasp-berries and sliced melons has been linked to pickers (Ackers et al., 1997; Lund and

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Snowden, 2000). For example, Ackers et al. (1997) report the probable connectionbetween hygiene of agricultural workers and an outbreak of cholera associated withsliced melon. Cross-contamination between crops may occur during handling andharvest and postharvest operations, and contamination with pathogens is possiblefrom handlers’ hands or from polluted wash water (Lund, 1992; ICMSF, 1998).Control measures include the use of clean water and sanitizers for washing fruitsand cleaning work surfaces, refrigeration of packing sheds and training of agricul-tural workers in good manufacturing and hygiene practices.

Contamination During Processing

The main sources of contamination during the processing of fresh-cut fruits and vege-tables are most probably the general factory environment and the processing equipment(Table 7.2). However, there is currently little information available documenting therisks of the contamination with individual organisms at this stage of production. Factoryworkers are also a possible source of contamination, but there is no supporting evidencein the literature. One study conducted by Garg et al. (1990) during the processing ofvegetables such as cabbage, lettuce and onions established that the shredders and slicerswere major sources of contamination. More information is needed about the microfloraof processing plants and the extent of contamination in this environment. Future studiesshould also investigate the role of biofilm formation in contamination of fresh-cutproducts. Recent studies have suggested that biofilm formation on processing equipmentmay provide contamination points (Carmichael et al., 1999).

Other non-vegetable ingredients may also be sources of contamination withmicroorganisms for fresh-cuts. In the case of low pH, dressed salads, processors maycombine fresh-cut fruits and vegetables with meat, chicken or seafood, thus increas-ing the range of potential spoilage microflora and the introduction of organisms ofpublic health significance. Christiansen and King (1971) examined meat-based salads,including ham, chicken and barbecue pork. Total counts for the ham salad rangedfrom 10

2

–10

6

counts/g salad, and counts for the other salads were slightly higher, upto 10

7

counts/g salad. The contribution of the meat to the microflora of the salads wasnot investigated. Little is known about the spoilage microflora of these salads.

FACTORS AFFECTING THE GROWTH OF MICROORGANISMS

Factors affecting microbial stability and quality of fresh-cut vegetables can be sim-plified into the four main categories listed below:

1. Intrinsic properties of the food—pH, water content, nutrients and protect-ing biological structures such as skin or cuticle

2. Processing factors—washing, blanching, cutting, shredding, packaging,conditions of temperature during the process and addition of preservatives

3. Extrinsic factors—storage temperature and use of modified atmospheres 4. Implicit properties of the microbial species—growth rate, temperature and

pH tolerance and interactions (Heard, 1999b)

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Table 7.3 relates these factors to the production of fruit and vegetables, fromthe farm through to the processed, packaged product, and the main factors are discussedbelow.

H

ANDLING

P

RACTICES

ON

THE

F

ARM

Environmental conditions such as temperature and rainfall, farm practices and thestandard of hygiene on the farm are all acknowledged as factors affecting themicrobial quality of fresh produce. During harvest and postharvest storage, microbialnumbers are believed to be influenced by the temperature, the hygiene of storageand transport facilities and the degree of damage of the produce during the harvest(Lund, 1992; Nguyen-The and Carlin, 1994; ICMSF, 1998; Brackett, 1999; Nguyen-The and Carlin, 2000). Surprisingly, although these factors are reported to be ofsignificance for the safe production of fresh produce, there is little documentedevidence of their effects on the individual microbial populations present throughoutgrowth, harvest and transport. The available literature is summarized below.

Water Quality

Water quality is an important factor influencing the microbial contamination of freshproduce during growth. Water is used for irrigation, washing, hand washing, coolingand for pesticide or foliar application (Pabrua, 1999). The methods used for irrigationcan significantly influence the extent of contamination. Sadovski et al. (1978), in astudy of the practice of wastewater irrigation, showed that contamination can be min-imized if covered drip irrigation is used rather than spray irrigation. Contaminationwith fecal coliforms was 38-fold higher on vegetables irrigated with sewage effluentwith an uncovered drip system than those irrigated with fresh water, but vegetablesirrigated with contaminated water through a drip system covered by soil werecontaminated with populations only 10-fold higher than the control. They also foundthat there was more risk of contamination if crops were watered with wastewaterjust prior to harvest rather than earlier in the growth cycle. Irrigation frequency mayinfluence the bacterial populations of crops during growth. Ludy et al. (1997)established that reduction of irrigation frequency from two to eight days reducedthe incidence of soft rot in broccoli heads from 30–15% in one year and from22–10% in another year. Use of contaminated water to prepare pesticides has alsobeen linked to outbreaks of foodborne disease. Recent studies have shown that thepathogens

Salmonella,

Shigella

and

E. coli

O157:H7 can survive and grow in pesti-cides (Coghlan, 2000) (Table 7.2). Washing the harvested crop can further increasemicrobial populations, particularly if wash water is not clean (ICMSF, 1998), andstorage and transport of crops in water can further encourage microbial growth.Segall and Dow (1973) reported contamination with species of

Erwinia

from pota-toes transported in water.

Good agricultural practices require knowledge of the source and safety of thewater and, if possible, knowledge of microbial populations, to prevent use of con-taminated sources. In many countries, health authorities have banned the use ofuntreated waters for irrigation. However, in many countries, there is no legislation

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TABLE 7.3Factors Affecting the Microbial Stability and Quality of Minimally Processed Vegetable Salads from Farm to Retail

Intrinsic Factors

pH• pH of salads vary depending on the type of vegetable used, e.g., tomatoes are low pH, lettuce

is at a neutral pH

Nutrient availability• mixed salads contain a wider variety of nutrients than single packs of vegetables• addition of non-vegetable ingredients such as dairy products, meat, seafood and chicken

provides fat and protein

Biological structure• the skin or cuticle of minimally processed vegetables is damaged and, therefore, not an obstacle

for microbial growth• the physiology of various vegetables, e.g., broccoli structure is complex, including tightly

packed florets and a stem with a waxy cuticle • the biochemical, physiological changes and interactions that occur in the salad during processing

and storage

Other factors• antimicrobial effects of vegetables, e.g., the antilisterial effect of carrots

Processing Factors

Farm practices• use of fertilizers• use of pesticides• contamination from handlers, farm animals and insects• damage during harvest• use of contaminated water• type of harvesting, i.e., manual or mechanical• condition of packing sheds and trucks

Washing• washing at the time of harvest may contaminate the vegetables with waterborne microorganisms• washing in the processing plant should reduce the microbial load (if the water is clean)

Temperature• temperature during harvest and trimming and washing on the farm• blanching or cooking of ingredients such as pasta and potato reduce or destroy vegetative

microbial cells• low temperature (0–5

°

C) during preparation, processing and storage limits the growth ofmicroorganisms

Other processes• processing operations such as chopping, shredding and slicing may contaminate the vegetables• assembly of salads may result in contamination from process workers and work surfaces• use of low pH dressings such as sour cream and mayonnaise lower salad pH • use of packaging to act as oxygen and water barriers and to prevent microbial contamination

(

continued

)

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controlling water quality in agriculture, and water remains a major source of con-tamination (Bryan, 1977; Beuchat, 1996, 1998; Pabrua, 1999).

Disinfectants

Washing fruits and vegetables in clean water can remove organisms from the surface,and the addition of a disinfectant (the use of chlorine, surfactants or acids such asperoxyacetic acid) can achieve additional 1–2 log reductions (Cherry, 1999). Beuchat(1998) reviewed processes for the surface decontamination of fruits and vegetables.Despite the lack of extensive scientific data, Beuchat (1998) makes a number ofconclusions about the efficacy of washing treatments and, in particular, the use ofdisinfectants. The efficacy of treatment varies with the following:

1. The type and pH of the disinfectant—disinfectants should be used withinthe pH range in which they are most active. For example, chlorine is mosteffective at a slightly acid pH, where the predominant and most effectivespecies is hypochlorous acid.

2. The time and type of contact—disinfectants such as chlorine are mosteffective within the first few seconds of treatment.

3. Water temperature—the temperature of the wash water should be higherthan that of the produce to avoid uptake of microbial cells by the tissue.

4. The properties of the produce—different surface structures can influenceinteractions with disinfectants.

5. The properties of the microorganisms—types of cells and stress influenceresponse to disinfectants. Resistance of pathogens to chlorine varies, andit is not known how effective disinfectants are in killing parasites andviruses on fruits and vegetables.

6. The level of contamination—heavily contaminated produce should bewashed twice, first to remove heavy soil and second to sanitize.

TABLE 7.3 Factors Affecting the Microbial Stability and Quality of Minimally Processed Vegetable Salads from Farm to Retail (Continued)

Extrinsic FactorsTemperature

• temperature fluctuations during transport and retailing

Modified atmospheres• modified atmosphere packaging influences survival of microorganisms during storage

Implicit Factors• competition between predominant microbial groups, e.g., lactic acid bacteria and pseudomonads• antagonistic relationships between microbial groups• synergism between microbial groups

Source: Adapted from Heard (1999b).

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Produce should also be dried after treatment to prevent growth of remainingorganisms. Beuchat (1998) also suggested that organic acids such as peroxyaceticacid and ozonation treatment showed good potential as disinfectants, but the condi-tions of use require more investigation.

Fertilizers

The choice of fertilizer can also influence the degree of contamination. The use ofinorganic fertilizers or composted, treated manure can reduce the risk of contami-nating crops with pathogens (ICMSF, 1998). Organic material can attach to outerleaves of produce, effectively enmeshing organisms on the surface. Bovine feceshave been reported as a source of E. coli O157:H7, and avian feces contain Salmo-nella spp. (Beuchat, 1996).

Damage

Injury of fruits and vegetables can occur during growth, for example, during hail-storms. Fruits and vegetables also often become injured during harvest resulting inrelease of nutrients and allowing entry of microorganisms to the internal tissues(ICMSF, 1998). Prior to injury or damage occurring, most microorganisms arepresent on the outside of fruits and vegetables and cannot enter the inner tissues dueto the cuticular layer covering the epidermis of aerial organs such as leaves, stemsand fruits (Nguyen-The and Carlin, 2000). Only true plant pathogens can invade thetissues of uninjured fruits and vegetables. Once the cuticular layer is broken, micro-organisms are exposed to cellular fluids and moisture, and microbial growth isencouraged. The release of juices containing sugars also encourages attack byinsects, further damaging the produce and allowing dissemination of microorgan-isms. Thus, hygienic handling conditions and cool temperatures during transport ofharvested produce to the processing plant are desirable to avoid excessive microbialcontamination (ICMSF, 1998; Heard, 1999b). Use of pesticides or use of organicfarming practices to deter insects is also desirable to reduce insect activity (Lundand Snowden, 2000).

Rainfall and Temperature

Warm, moist conditions during harvest are known to increase the overall microbialload of crops such as tomatoes (Senter et al., 1985). The rainfall during growth ofvegetables has also been shown to affect the microbial population. Mundt and co-workers (1967, 1970) studied the numbers and types of lactic acid bacteria onvegetables and reported that populations of these organisms are likely to be low anddifficult to recover in conditions of near drought. Numbers of the predominantspecies, Leuconostoc mesenteroides, reached as high as 104 cfu/g on green vegetableswhen rainfall was more abundant.

Temperature is also an important factor influencing microbial numbers duringpostharvest handling. Splittstoesser (1970) observed that fresh vegetables, such asspinach and peas, harvested and transported to the processor under warm, humid

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conditions carried up to 107cfu/g total bacteria. Rapid transport of produce undercool conditions should be encouraged to minimize microbial growth.

The full extent of the influence of farm practices on the quality of fruits andvegetables used for fresh-cuts and the effect on the final processed product is yetto be determined. However, certain control measures may be taken to reduce con-tamination, including use of refrigeration and clean water during postharvest oper-ations. Detailed ecological investigation is required for us to determine the mostimportant microbial species present and the most important influencing factors. Withevidence for the importance of hygienic practices on the farm, we can educate andtrain farm workers in the safe production of fresh produce for all applications.

CONDITIONS DURING PROCESSING/PACKAGING

What are the factors influencing the survival and growth of the microflora of freshproduce during the processing stages? The predominant species at the time of harvestand transport will not necessarily dominate during processing, and it may not be theseoriginal species that cause the spoilage symptoms (Nguyen-The and Prunier, 1989;Bennick et al., 1998) or cause outbreaks of foodborne disease (Beuchat, 1998). Opti-mization of conditions to extend the shelf life of these products is the main priorityof processors, as microbial spoilage is of major commercial significance and willcontinue to be so as the market expands worldwide. Currently, the main hurdles appliedto control microbial growth are the use of low-temperature and modified atmospheres.Unfortunately, the effects of processing, low-temperature storage and modified atmo-sphere storage on individual species or microbial groups present on the raw produceare yet to be studied. We also lack information about biochemical reactions that mayassist in the development of microorganisms and interactions between species thatmay occur during processing as the structures and intrinsic properties of the produceare altered. The following section mainly examines the processing factors and condi-tions that influence the growth of microorganisms in salads, as reported in the literature.

Processes

On arrival at the processing plant, raw fruits and vegetables are trimmed and peeled.Trimming processes are the first steps taken toward reducing microbial load of theproduce by removing the most heavily contaminated outer layers. Decay of leafyvegetables such as endive occurs more frequently on the outer green leaves than onthe inner yellow leaves (Carlin et al., 1995). However, the actual process of removalmay result in contamination of the edible portion of the fruit or vegetable, thusincreasing the risk of microbial contamination during subsequent processing steps(Garg et al., 1990; Beuchat, 1998). Thus, washing after trimming and cutting is nec-essary to remove microbes (Sinigaglia et al., 1999). Washing may not always removemicrobial cells, and processors should be aware that cells may attach to the tissuesof produce. For example, Babic et al. (1996), while examining the surface of processedspinach leaves using scanning electron microscopy, found that microorganismswere embedded in damaged spinach cells or in cells adjacent to the damaged tissue.Garg et al. (1990) investigated the effect of processing on the total microbial counts of

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Microbiology of Fresh-cut Produce 203

vegetables including cabbage, lettuce and onions. The shredders and slicers were foundto be major factors influencing the level of contamination. The aerobic plate countof lettuce increased from 1.8 × 104 cfu/g to 140 × 104 cfu/g after shredding. Similarly,the aerobic plate count of onions increased from 4.0 × 103 to 1.2 × 105 cfu/g duringslicing. More recently, Barry-Ryan and O’Beirne (1998) determined that the methodof slicing also influences the microbial load of vegetables. Microbial counts of carrotslices sliced using a razor and a slicing machine were compared (both blunt andsharp blades were used in the machine). Total microbial counts, coliforms, lacticacid bacteria and yeast and mold counts were all higher on slices prepared with themachine than slices cut with a razor. For example, after the carrots had been storedfor one day, the total aerobic counts on razor-sliced carrots were 5.77 log10/g, andthe counts on carrots cut with a blunt blade were 6.27 log10/g. However, the effectsof the machine blade, blunt or sharp, were only significant for population of Pseudomo-nas and coliforms. Advances in size reduction technology may be used in the futureto reduce contamination of vegetables. For example, high-pressure water jets and CO2

lasers may be used as tools for cutting (Sanguansri, 1997). These methods result incleaner cuts and minimum dust generation, and the CO2 laser partially sterilizes thecut surface. The effect of such technologies on the microbial ecology of cut vege-tables is yet to be determined.

Sanitation

How important are sanitation and the application of hygienic principles during theprocessing of fruits and vegetables? All handlers of produce in the processingenvironment must ensure the highest level of hygiene. Cleaning and sanitation ofequipment and processing surfaces is also of utmost importance to prevent buildupof organic residues that might encourage growth of microorganisms and formationof microcolonies or biofilms (Brackett, 1989; Carmichael et al., 1999). The signif-icance of biofilm formation in processing environments is yet to be determined.Other processes, including washing, may contribute to the spoilage microflora ofthe processed vegetables, especially where recycled water is used. Buildup of organicresidues in the water can result in growth of spoilage microorganisms, increasingthe potential for contamination (Brackett, 1992).

Washing fruits and vegetables with water alone only achieves a small (1 log)reduction in microbial numbers (Nguyen-The and Carlin, 1994). Chlorinated water(100–200 mg/L) is widely used for washing and sanitizing minimally processedfruits and vegetables (Ahvenainen, 1996), although the effect of chlorine on micro-organisms attached to surfaces is limited. As mentioned previously, the efficacy ofdisinfection treatments varies with the concentration of the disinfectant used, timetaken to process vegetables and temperature during processing and level of contam-ination of the produce with organic matter and microbial cells. Table 7.4 summarizesa number of reports of commonly used disinfectants and their efficacy for reducingpopulations of microorganisms on processed fruits and vegetables. Most workershave concentrated their research efforts on the reduction of total microbial popula-tions and L. monocytogenes in salads. It is possible to achieve a 3 log reduction intotal count on lettuce using 300 mg/l-1 sodium hypochlorite (Garg et al., 1990),

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TABLE 7.4Effect of Common Disinfecting Treatments of Microbial Populations on Fresh-cut Fruit and Vegetable Products (at the Time of Treatment)

Disinfecting Treatment ProductEffect on Microbial

Count Ref.Chlorine (300 mg/L free) pH 9.4

Lettuce leaves 0.7% of the unwashed count (approximately 107 cfu/g)

Adams et al. (1989)

Chlorine (100 mg/L free) pH 5

Lettuce leaves 6.22 log reduction Adams et al. (1989)

• sulphuric acid 5.83 log reduction• acetic acid 5.58 log reduction• citric acid 5.77 log reduction• lactic acid 5.6 log reduction• propionic acid 5.6 log reduction

Chlorine (210–289 µg/ml) water

Chopped tomatoes No reduction in counts of L. monocytogenes or aerobic plate counts when compared to untreated product

Beuchat and Brackett (1991)

Chlorine 200 ppm (10 min) Lettuce 1.79 log reduction in Salmonella populations (compared to untreated)

Beuchat et al. (1998)

2.48 log reduction in E. coli O157:H7 population (compared to untreated)

0.33 log reduction in aerobic mesophile population (compared to untreated)

Cl2 wash (concentration not specified, no treatment control)

Cantaloupe (fresh cut)

Approximately 0.12 log reduction in fluorescent pseudomonads when compared to untreated product

Sapers and Simmons (1998)

Cl2 wash (concentration not specified, no treatment control)

Zucchini (fresh cut) Approximately 1.5 log reduction in fluorescent pseudomonads when compared to untreated product

Sapers and Simmons (1998)

Chlorine wash (100 µg/mL) • warm wash at 47°C• chilled wash at 4°C

Shredded lettuce 3 log reduction after warm wash

1 log reduction after chilled wash

Delaquis et al. (1999)

Chlorine dioxide (5 mg/l, 10 min, 40°C, pH 7.4)

Shredded lettuce 1.1 log reduction of L. monocytogenes

Zhang and Farber (1996)

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Microbiology of Fresh-cut Produce 205

although commercially, chlorine concentrations are generally lower, 100–200 ppm.At these concentrations, a 1–2 log reduction in total aerobic count would be expectedon processed lettuce and endive ( Adams et al., 1989; Beuchat and Brackett, 1990b).The effect of chlorine on several pathogens has been established on only a limited

TABLE 7.4Effect of Common Disinfecting Treatments of Microbial Populations on Fresh-cut Fruit and Vegetable Products (at the Time of Treatment) (Continued)

Disinfecting Treatment ProductEffect on Microbial

Count Ref.Dichloroisocyanurate (equivalent to 40–320 ppm free chlorine)

Vegetables 1.69–2.42 log reduction compared to wash with water alone

Nicholl and Prendergast (1998)

Electrolyzed water (20 ppm available chlorine)

Fresh-cut vegetables

Log reduction (compared to untreated)

Izumi (1999)

0.3• carrot 1.8• spinach 0.4• bell pepper 0.2• Japanese

radish potato0.4

H2O2 (no treatment controls)

Cantaloupe (fresh-cut)

Approximately 0.68 log reduction in fluorescent pseudomonads when compared to untreated product

Sapers and Simmons (1998)

H2O2 (water and no treatment controls)

Mushrooms (fresh cut)

Approximately 1 log reduction in fluorescent pseudomonads when compared to untreated or water-washed product

Sapers and Simmons (1998)

H2O2 (no treatment controls)

Zucchini (fresh cut) Approximately 1 log reduction in fluorescent pseudomonads when compared to untreated product

Sapers and Simmons (1998)

H2O2 (5%) Fresh-cut fruits and vegetables

3 log kills Cherry (1999)

Isothiocyanate 400 µL allyl isothiocyanate (2–4 days treatment)

Iceberg lettuce Up to 8 log reduction of E. coli O157:H7

Lin et al. (2000)

Ozone (1.3 mM ozone, injected at 1.5 L/min for 3 min)

Shredded lettuce 2 log reduction in total count

Kim et al. (1999)

Peroxyacetic acid (200 ppm)

Fresh-cut fruits and vegetables

2 log kills Cherry (1999)

Count taken immediately after treatment.

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range of processed products. Zhang and Farber (1996) reported reduction ofL. monocytogenes on shredded lettuce and cabbage. They achieved a 1.3–1.7 log10cfu/g on lettuce and 0.9–1.2 log10 cfu/g on cabbage after treatment with 200 ppmchlorine for 10 min. Beuchat et al. (1998) measured the reduction of E.coli O157:H7,Salmonella and L. monocytogenes on whole and cut lettuce sprayed and rinsed withchlorine solutions of 200 ppm. Compared with control samples that were washedin water, reductions of only 1 log cfu/g were achieved by treatment with chlorine.Further investigation is needed of the effect of disinfectants on pathogenic specieson a wider range of products.

Recent controversy over the safety and effectiveness of chlorine (Sapers andSimmons, 1998) has encouraged interest in alternative sanitizers, including peroxy-acetic acid, chlorine dioxide, ozone, trisodium phosphate and hydrogen peroxide(Beuchat, 1998; Sapers and Simmons, 1998; Xu, 1999). Detailed studies are neededto determine the effectiveness of these compounds for reducing the initial load ofthe produce and also to determine their role in shelf life extension of the cut produce.

Temperature

Temperature control is commonly used to prevent or minimize the microbial spoilageof foods. Although refrigeration of fruits and vegetables does not completely inhibitmicroorganisms, it reduces the growth rates of some spoilage organisms and food-borne pathogens (Nguyen-The and Carlin, 2000). However, we need to establish towhat extent temperature control is important during minimal processing of vegeta-bles. The key issue is how does the temperature profile of the produce from receivalat the factory and throughout processing influence the growth of individual species’microbial interactions? Is it crucial for the quality of the final product to refrigerateproduce immediately after harvest and to maintain refrigeration conditions at allstages of processing? Ideally, to answer these questions, we should study the micro-bial ecology of vegetables and the effect of temperature on this ecology throughoutall stages of the production chain. Unfortunately, there are no reports of the tem-perature effect during processing, but most processors maintain lower than ambienttemperatures during processing operations. From the time of arrival, processorsattempt to reduce the temperature of raw fruits and vegetables to refrigerationtemperatures, 5°C. The environment in which trimming and peeling processes areperformed is maintained at around 10–15°C, and wash water is generally refriger-ated. After processing, fresh-cut products are cooled to 2–5°C (Ahvenainen, 1996).

There are several publications investigating the effect of storage temperature onmicrobial development in fresh-cut products. Storage at refrigeration temperaturesgenerally selects for growth of psychrotrophic organisms (Nguyen-The and Carlin,1994, 2000), even though mesophilic and psychrotrophic counts may be of similarmagnitude prior to processing (Garg et al., 1990). Refrigeration prevents the growthof Erwinia spp. and several spoilage fungi, including Fusarium and Phytophthera spp.but does not prevent the growth of Pseudomonas fluorescens (Lund, 1983; Nguyen-The and Carlin, 2000). Mesophilic organisms may also grow at refrigeration tem-peratures, at reduced growth rates (Marth, 1998). Manvell and Ackland (1986) used thepresence of lactic acid in salads as an indicator of temperature abuse. Gram-negative

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Microbiology of Fresh-cut Produce 207

organisms were found to predominate in mixed salads containing leafy vegetablesand in carrots at 7°C, whereas lactic acid bacteria could proliferate in salads at 30°C.Growth of mesophilic organisms is reduced on leafy vegetables as the storage tem-perature decreases (2°C) (Bolin et al., 1977; Carlin and Nguyen-The, 1989; Magnussonet al., 1990; Beuchat and Brackett, 1990b), presumably because the temperature isbelow optimum growth temperature. However, spoilage of salads at low temperaturesmay not be the result of psychrotophic bacteria alone. For example, Vescovo et al.(1996) observed a 2 log increase in mesophilic counts in mixed salads stored at 8°Cfor six days. King et al. (1991) investigated the effect of temperature on bacterialgrowth on lettuce. Bacterial counts increased significantly on lettuces stored at 2, 5and 7°C, although growth rates were similar at all temperatures. It was assumed thatthe bacteria were growing below their optima at all storage temperatures used in thestudy. Growth rates of lactic acid bacteria on shredded carrots were found to decreaseas storage temperatures were lowered (Carlin et al., 1990; Kakiomenou et al., 1996).Similarly, Guerzoni et al. (1996) showed that processing time and temperaturedirectly influenced the proliferation of contaminants on lettuce. Lettuces treated withchlorine solution (165 ppm) were stored at either 5 or 12°C for six hours aftertreatment, then subsequently stored at 5°C for up to 10 days. Lettuces exposed tothe higher temperature were more heavily contaminated with bacteria (107cfu/gcoliforms) than those stored at 5°C (105 cfu/g coliforms).

The effect of temperature on the survival and activity of pathogens on fresh-cutproduce is not widely reported in the literature, and most reports focus on L. mono-cytogenes. Storage of processed fruits and vegetables at refrigeration temperaturesmay deter the growth of mesophilic pathogens but will not necessarily prevent survivaland growth of L. monocytogenes or Aeromonas hydrophila. However, refrigeratedstorage may reduce the growth rate of these pathogens, thus reducing the risk of theirdevelopment during the storage life of fresh-cut products. Listeria monocytogeneswas reported to survive on shredded lettuce at 5°C, although no significant growthwas recorded for up to eight days of storage (Beuchat and Brackett, 1990b). Byincreasing storage temperature to 10°C, growth was significant after three days ofstorage, and at 10 days, 108–109 cfu/g were recorded. Similarly, Aytaç and Gorris(1994), Carlin and Nguyen-The (1994) and Jacxsens et al. (1999) reported slowgrowth of L. monocytogenes on a range of lettuce and chicory endives at temperaturesunder 10°C. Kallander et al. (1991) found that L. monocytogenes grew rapidly (1 logincrease by day two) on cabbage for the first few days of storage at 5°C, but numbersdecreased after six days, at which time the cabbage was spoiled and pH was reduced.Aeromonas was reported by Aytaç and Gorris (1994) and Jacxsens et al. (1999) toexhibit a higher growth rate than Listeria, increasing on chicory endive by 4 logsafter seven days of storage at 6.5°C. Clostridium botulinum can also survive atrefrigeration temperatures, although toxin is generally not produced if the temperatureis lower than 15°C (Petran et al., 1995). Enteric viruses can also survive in processedvegetables at 40°C. Poliovirus decreased by only 1 log cycle on cut vegetables storedat 4°C for 10 days (Croci et al., 1991). There are limited reports of survival of otherpathogens in refrigerated processed fruits and vegetables. The main risks from patho-gens such as Staphylococcus aureus and Salmonella spp. arise from products thatmay undergo temperature abuse. These organisms are known to survive on vegetables,

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including tomatoes and mushrooms, stored at 20–35°C. Further studies of the effectsof temperature fluctuations throughout processing and storage of fresh-cut productswill allow us to better assess the risks of the growth of pathogens.

There are few reports investigating the effect of temperature on the spoilage oflow pH, dressed salads. King et al. (1976) studied the effect of low temperature onthe microflora of coleslaw dressed with sour cream and mayonnaise. Deteriorationof salad quality was observed for coleslaw stored at two temperatures, 7 and 14°C.At 14°C, the total microbial count increased logarithmically during storage, whileat 7°C, the microbial count gradually decreased. No spoilage organisms were detectedin the coleslaw stored at the lower temperature, and it was assumed that deteriorationof the salads was due to physiological changes occurring in the cabbage tissue.Brocklehurst et al. (1983) studied the microflora of coleslaw stored at 5 and 10°C.Coleslaw dressed with mayonnaise supported growth of Saccharomyces exiguusduring storage at 5°C and Saccharomyces dairensis during storage at 10°C. Lacticacid bacteria could not grow during storage at 5 or 10°C.

pH

Lowering the pH of foods to within the range 3.0–5.0 restricts the types of micro-organisms able to grow, thus reducing the risk of spoilage or the growth of organismsof public health significance. Traditionally, this has been achieved by fermentation oraddition of acidic ingredients. We are currently unsure of the effect of minimal pro-cessing on the pH of fruits and vegetables. Can the pH be altered during minimalprocessing of fruits and vegetables to create hurdles for spoilage organisms andmicroorganisms of public health significance? In the case of many fruits, the pHmay already be sufficiently low to deter spoilage bacteria. Can the acidity of fruitsbe relied on to reduce the risk of spoilage of growth of pathogens in mixed fresh-cut products? The influence of pH changes during processing on the microflorashould also be investigated. Only a few studies have reported pH changes forminimally processed vegetables, including dressed salads. King et al. (1991) notedthat pH of lettuce increased during storage, concurrently with an increase in bacterialpopulation that was predominantly Gram-negative. A decrease in pH was observedfor vegetables such as shredded carrots, where the predominant flora is lactic acidbacteria (Kakiomenou et al., 1996). Growth of spoilage bacteria, presumably lacticacid bacteria, on shredded cabbage and the resulting reduction in pH prevented theproliferation of L. monocytogenes (Kallander et al., 1991). Marchetti et al. (1992)observed that development of microbial flora on minimally processed salads wasnot simply related to pH and the presence of organic acids. They concluded thatspoilage patterns were also related to characteristics of the raw materials. This furtheremphasizes the need to investigate interactions between pH and other factors affect-ing the shelf life of ready-to-eat salads.

Fresh-cuts may be dressed with acidic sauces or creams such as mayonnaise orsour cream to lower the pH. This creates a selective environment that favors growthof acid-tolerant microorganisms. The predominant flora of dressed, low-pH saladsare bacteria such as acid-tolerant lactobacilli and yeasts (Hildebrandt et al., 1989;Hunter et al., 1994). Growth of other microorganisms is either inhibited by the acetic

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Microbiology of Fresh-cut Produce 209

acid present in mayonnaise or by acids produced by the sour cream culture or bycompetition, in the case of sour cream dressings. For example, King et al. (1976)concluded that the microflora of coleslaw prepared with sour cream was the sameas that of the cream, replacing the cabbage microorganisms.

Brocklehurst et al. (1983) and Brocklehurst and Lund (1994) studied the effectof pH on spoilage of salads. They reported that acetic acid in mayonnaise was inhibitoryto lactic acid bacteria and the spoilage yeasts, S. dairensis and S. exiguus, in potatosalads and coleslaw. However, the addition of salad ingredients reduced inhibitoryeffects of mayonnaise. It was hypothesized that salad ingredients absorbed aceticacid, resulting in an overall increase in salad pH. The combined hurdles of low tem-perature and low pH during storage also influenced growth. The acid environmentis more inhibitive at refrigeration temperatures (5°C) than at higher temperatures(10°C), emphasizing the importance of controlling temperature and pH during pro-cessing. Fresh-cut produce are now mixed with ingredients such as pasta and a rangeof salads based on low-fat and sour cream dressings and low-acid dressings that arenow available. The significance of using less acid and the changes to pH duringproduction and storage of salads should be investigated, focusing on the survival ofindividual microbial species.

Packaging

The final stage in production of fresh-cut vegetables is packaging. The packageprovides protection for the fresh-cut product from damage and further contaminationwith microorganisms. The use of controlled and modified atmosphere packagingalso provides, to some extent, a hurdle against the growth of the remaining spoilagemicroflora and foodborne pathogens (Phillips, 1996). However, the microbial ecol-ogy of packaged fresh-cut products is poorly understood. Investigation is neededof the influence of packaging methods on the growth of individual species, howpackaging and controlled atmospheres influence interactions between species and,ultimately, how they influence the microbial quality and safety of the products. fresh-cut fruits and vegetables are either packaged unsealed, in the presence of air or undera modified atmosphere (MA) (Ngyuen-The and Carlin, 1994). If a package is sealedunder air, the MA results from the respiration of the product. Alternatively, the packagemay be filled with a specific gas mixture, for example, 5–10% CO2 and 2–5% O2

as is used to extend shelf life of whole vegetables (King and Bolin, 1989). Furtherreduction of oxygen or increase in carbon dioxide concentrations may be deleteriousto the physiology of the product, for example, browning reactions may be encouraged(Kader et al., 1989; Phillips, 1996).

The influence of MA packaging on the physiology and microbiology of fruits andvegetables has been reviewed (Nguyen-The and Carlin, 1994, 2000; Phillips, 1996;Bennick et al., 1996, 1998; Kader and Watkins, 2000), and it is clear that the benefitsof MA cannot be explained solely by a reduction in total microbial load. Other influ-encing factors include the packaging material used, the use of microperforations in thematerial, relative humidity during storage, time prior to packaging, temperature ofstorage, type of produce and type and number of microorganisms present and thenutrients available to support microbial growth. A combination of low-temperature

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storage and MA packaging is recommended to enhance quality of fresh-cut fruits andvegetables and to extend shelf life. There have been few attempts to relate the effectof packaging on the individual spoilage organisms and on the overall acceptability ofvegetable products throughout storage. However, a number of studies investigated theuse of modified atmosphere packaging and the effect of increasing CO2 concentrationson the survival of L. monocytogenes and other pathogens (Table 7.5).

The effect of modified atmosphere packaging on the microbial ecology andorganoleptic qualities of lettuce has been studied by Magnuson et al. (1990) andKing et al. (1991) (Table 7.5). Modified atmosphere was created passively in bothstudies. The major difference between lettuces stored unsealed and sealed in bagswas the influence of the MAP on the microflora. A wider variety of microorganismsgrew on unsealed packaged lettuce than on the packaged lettuce. Although bacteriainitiated spoilage, the yeasts Pichia fermentans and Torulaspora delbrueckii devel-oped as the conditions became anaerobic in the sealed bags. Higher microbial counts,including Pseudomonas counts, were reported in unpackaged and shrink-wrappedcut broccoli than in gas-packed samples by Brackett (1989). However, populationsof Enterobacteriaceae were similar regardless of packing treatment. Gas-packedsamples remained fresh after six weeks of storage, unlike other treatments in whichthe broccoli was spoiled, suggesting that modified atmosphere packaging may delaythe spoilage of a product. Kakiomenou et al. (1996) observed that the onset ofspoilage of shredded carrots by lactic acid bacteria was delayed in carrots stored at5% CO2. Lactic acid bacteria predominated in packaged and unpacked samples,although organic acid production was higher under MA packaging, suggestinggreater metabolic activity under these conditions.

Bennick et al. (1996, 1998) presented a detailed ecological study of the effectof MA packaging on the predominant flora of chicory and mung bean sprouts. Thenumbers and types of organisms and the growth rates of these organisms wererecorded during storage. Overall, MA did not influence maximum population den-sities, but the increase in CO2 in packages resulted in the reduction of maximumspecific growth rates, particularly for Pseudomonas spp. Changes in predominanceof microbial species were specific to different vegetables, suggesting specific effectsof the MA on microorganisms, thus reinforcing the need to investigate factorsaffecting the growth of individual organisms present on vegetables.

Packaging under modified atmosphere generally does not inhibit the growth ofL. monocytogenes, although other factors influence its survival (Beuchat and Brackett,1990b; Kallander et al., 1991; Aytaç and Gorris, 1994; Carlin et al., 1996; Jacxsenset al., 1999). Listeria monocytogenes grows well on lettuce and chicory endive undermodified atmosphere. Carlin et al. (1996) observed stored chicory endive under MAincreasing CO2 concentration as high as 50%. They observed that Listeria grewbetter as the CO2 increased. Kallander et al. (1991) showed that temperature affectedthe growth of L. monocytogenes under MA conditions. At 5°C, no difference wasobserved in growth of the organisms on shredded cabbage, but at 25°C, initial growthwas reduced, and a rapid decline was observed after six days of growth. This wasmost probably due to the excessive growth of spoilage flora under the MA conditions.Jacxsens et al. (1999) observed the effect of an equilibrium atmosphere of 2–3%O2:3% CO2:94–96% N2 on the growth of L. monocytogenes and Aeromonas spp. on

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TABLE 7.5Influence of Packaging on the Growth of Microorganisms in Salads/Vegetables

Salads/ Vegetables Packaging Treatment Growth of Spoilage Microorganisms Spoilage Characteristics Ref.Broccoli 1. No package

2. Shrink-wrap3. MAP pouches (5% O2, 10%

CO2, 85% N2)

Higher total counts in treatment 1 than in other treatments. No differences between counts of Enterobacteriaceae. Pseudomonas counts were higher in treatment 1 than in other treatments. Coryneform bacteria predominated in shrink-wrap packages. MAP broccoli had a longer shelf life than in other treatments.

Broccoli in no packaging was spoiled after six weeks storage—yellowing, slimes, wet lesions.No spoilage symptoms in other treatments at six weeks.

Brackett (1989)

Broccoli florets 1. No package2. Packaged

Aerobic plate count and coliform counts were consistently higher in unpackaged than in packaged during storage at 8°C for seven days.

Moisture loss in unpackaged florets, less crisp than packaged florets.

Mohd-Som et al. (1994)

Shredded cabbage

Packages1. Normal atmosphere2. Modified atmosphere (70%

CO2, 30% N2)

After 20 days storage at 5°C, population of Listeria monocytogenes gradually increased up to 13 days followed by a rapid decline, 2.0 log, under normal atmosphere conditions. Under modifed atmosphere, there was an approximate 1.0 log increase.

— Kallander et al. (1991)

Chicory/endive Packaged1. No vacuum2. Half vacuum

Aeromonas hydrophila population increases 4 log units, seven days storage with no vacuum, gradual loss of viability under vacuum.Listeria monocytogenes maintained viability in both packages (approximate 2 log increase under vacuum).

— Aytaç and Gorris (1994)

(continued)

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TABLE 7.5 Influence of Packaging on the Growth of Microorganisms in Salads/Vegetables (Continued)

Salads/ Vegetables Packaging Treatment Growth of Spoilage Microorganisms Spoilage Characteristics Ref.Chicory/endive 1. Control—21% O2, 0% CO2,

78% N2

Control—50% higher maximum growth rate of the predominant spoilage organisms than in the MAP treatment.

Control—dicoloration and softening, rotting.

Bennick et al. (1996)

2. MAP—1.5% O2, 20% CO2, 78% N2

MAP—Predominant organisms include Ps. fluorescens, Ps. corrugata, Ps. putida, species from the family Enterobacteriaceae.

MAP—good appearance, acceptable quality after 13 days of storage.

Shredded chicory/endive

1. Air2. Equilibrium modified

atmosphere (EMA) (2–3% O2, 3% CO2, 94–96% N2)

Changes (increase) in microbial populations over 6–7 days storage (7°C)

Total count: Air—2.36, EMA—2.52Aeromonas: Air—0.5, EMA—2.5Listeria monocytogenes: Air and EMA—0-0.1

Less enzymic discoloration, longer retention of rigidity, beneficial to physiological state of the vegetable in EMA

Jacxsens et al. (1999)

Lettuce Polyethylene (38 µm) bags1. Unsealed2. Sealed

Initial spoilage of all samples by bacteria. Wider variety of yeast species in unsealed bags. Growth of fermentative yeasts (Pichia fermentans and Torulaspora delbrueckii) was encouraged.

Dicoloration and softening. (Lettuce stored for 11 weeks.)

Magnuson et al. (1990)

Lettuce Polyethylene bags1. Sealed2. Unsealed

Slower bacterial growth in sealed bags, wider variety of yeast species in unsealed bags

Discoloration and softening King et al. (1991)

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Shredded iceberg lettuce

1. Air2. Equilibrium modified

atmosphere (EMA) (2–3% O2, 3% CO2, 94–96% N2)

Changes (increase) in microbial populations over six to seven days storage (7°C)

Total count: Air—2.45, EMA—1.16Aeromonas: Air—0.2, EMA— −0.1Listeria monocytogenes: Air— −1.5

Less enzymic discoloration, longer retention of rigidity, beneficial to physiological state of the vegetable in EMA.

Jacxsens et al. (1999)

Mixed vegetable salad

Plastic filmInitial headspace1. Air2. MA—10.5% CO2, 2.25% O2,

87.25% N2

Total microbial counts—no difference between packaging treatments.

Acceptable in MAP after 10 days. Priepke et al. (1971)

Shredded carrots Bags1. Air2. MAP—5% CO2, 95% N2

3. Polyethylene (60 µm)—4.9% CO2, 2.1% O2, 93% N2

Lactic acid bacteria predominated in all treatments. Organic acid production increased for carrots stored under MAP.

Better texture, color and odor under MAP. Delayed spoilage.

Kakiomenou et al. (1996)

Source: Adapted from Heard (1999b).

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a range of vegetables, including shredded lettuce, chicory endive, Brussels sproutsand carrots. Brussels sprouts and carrots were found to inhibit the growth of thepathogens, but both organisms grew under the MA conditions. There is also a generalconcern that anaerobic conditions of MA-packaged fresh-cuts will support thegrowth of anaerobic clostridia, in particular, Cl. botulinum (Nguyen-The and Carlin,2000); however, there is little information available concerning the risks.

CONDITIONS DURING RETAILING

There is no scientific evidence documenting the influence of retail conditions on themicrobial quality of fresh-cut products. Nevertheless, if we apply the main factorsinfluencing the microbial ecology of foods as summarized in Table 7.3, the maininfluencing factors can be predicted. The shelf life and microbial quality of fresh-cut products during transport and retailing are dependent on the following:

1. Method of transport—refrigerated transport should be used to maintaintemperatures below 5°C.

2. Time—transport and loading times should be minimized, e.g., temperaturefluctuations occur when products are left on loading docks or in storagerooms at supermarkets.

3. Temperature of storage cabinets—refrigerated cabinets should be main-tained at temperatures below 5°C.

The final factor affecting the quality during retailing is handling by the consumer.Fresh-cut products are highly perishable products and should be stored under refrig-eration after purchase. Education on proper food handling practices should be pro-vided for supermarket handlers and consumers.

OTHER FACTORS

The intrinsic properties of some vegetables and implicit characteristics of the micro-organisms may influence development of the microflora during the production andstorage of fresh-cut produce. For example, carrots are reported to exhibit antiliste-rial effects (Beuchat and Brackett, 1990a) and may be used in combination withother ingredients as an extra hurdle to prevent growth of L. monocytogenes. Marchettiet al. (1992) observed that red chicory and carrot juices exhibited antimicrobialactivity against Ps. fluorescens and L. monocytogenes. Background microflora ofspinach, endive and lettuce have also been reported to restrict the growth of Listeriaspp. (Carlin et al., 1996; Babic et al., 1997; Francis and O’Beirne, 1998). For example,Enterobacter spp. were found to compete with L. innocua on processed lettuce(Francis and O’Beirne, 1998). Listeria monocytogenes may also be inhibited in foodsby Ps. fluorescens and lactic acid bacteria. Pseudomonas spp. produce siderophoresto bind essential nutrients, and lactic acid bacteria may produce antimicrobial bac-teriocins in addition to lactic acid and hydrogen peroxide (Harris et al., 1989).

Finally, it is essential to ask if there are interactions in fresh-cut products betweenthe indigenous spoilage flora such as the pseudomonads and foodborne pathogens.It has been suggested that removal of the indigenous spoilage microflora of fresh

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foods such as fruits and vegetables “opens the door” for pathogens and allows themto proliferate. Preliminary investigations have indicated that biofilms occur on veg-etable leaves (Carmichael et al., 1999). Biofilms formed by nonpathogenic speciesmay form a natural barrier to prevent attachment of pathogens to surfaces of fruitsand vegetables. Further study is required to better understand leaf ecology andinteractions. Seo and Frank (1999) investigated the colonization of lettuce leaves byE. coli O157:H7 and Ps. fluorescens. The use of confocal scanning laser microscopyand dual staining techniques enabled detection of the different organisms on the leafsurface. The pseudomonad preferentially adhered to the intact leaf surfaces, whilethe pathogen colonized cut edges and stomata. Interestingly, the pathogen did notadhere to the biofilm formed by the pseudomonad on leaf surfaces.

MICROBIAL SPOILAGE OF FRESH-CUT PRODUCTS

EFFECT OF MICROBIAL GROWTH ON THE QUALITY AND SHELF LIFE OF FRESH-CUT SALADS

Spoilage symptoms appear as microbial numbers increase, and as a result, qualityis reduced and shelf life shortened. However, the degree of spoilage does not alwayscorrelate with large total populations (Nguyen-The and Carlin, 2000). One example,reported by Nguyen-The and Prunier (1989), was the spoilage of chicory endivesalad by Pseudomonas marginalis. After initial investigation, it was concluded thatdeterioration of the product with this organism occurred because it was the predom-inant species. However, treatment of the vegetable with pure cultures of other species(108 cfu/g leaves) failed to induce spoilage. As described in the previous section,environmental factors, the types of fruits or vegetables and the types of organismspresent determine what type of spoilage occurs and how quickly the quality of theproduct deteriorates. For example, pectinolytic pseudomonads present on chicoryendive leaves will cause spoilage (Nguyen-The and Prunier, 1989). However, carrotsstored at 10°C and contaminated with equal numbers of pseudomonads and lacticacid bacteria exhibit spoilage symptoms of the latter (Carlin et al., 1989). Unlikewhole vegetables, fresh-cuts are not spoiled by the soft rot bacteria Erwinia, sug-gesting that either the environment does not encourage their growth or that otherbacteria outgrow these species (Nguyen-The and Carlin, 2000).

Despite these observations, our knowledge of the spoilage patterns of fresh-cutproducts is limited. Most manufacturers base shelf life predictions on total microbialcounts or groups of microorganisms and observation of the associated spoilagedefects as well as the extent of enzymatic degradation of the tissues. We do not fullyunderstand the microbial interactions occurring or the factors influencing spoilageby individual species in the finished product, and further study is required.

SPOILAGE CHARACTERISTICS

Spoilage of fresh-cut vegetables by bacteria is characterized by brown discoloration,production of off-odors, loss of texture and, to a lesser extent, soft rot. Fruit productsundergo fermentative spoilage by lactic acid bacteria or yeasts, resulting in the

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production of acids, alcohol and CO2, although pseudomonads may spoil less acidicfruits such as cantaloupe. Lipase activity and utilization of amino acids can alter theflavor of fruits, resulting in a loss of quality. The main types of spoilage responsiblefor deterioration of quality and the mechanisms of spoilage are summarized as follows:

1. Soft rots—maceration of the vegetable tissue caused by enzymatic deg-radation of the plant cell wall by pectinolytic enzymes (Liao and Wells,1987)

2. Formation of off-odors and off-flavors—activity of lipolytic and proteolytcenzymes and fermentation reactions (Kato et al., 1989; Wyllie and Leach,1992; Zhuang et al., 1994, Lamikanra et al., 2000)

3. Wilting—brought on by vascular infections (Schroth et al., 1992)4. Brown discoloration—polyphenol oxidase activity of the microflora may

contribute to browning (Padaga et al., 1999)5. Fermentative spoilage—fermentation of carbohydrates to produce acid,

gas or alcohol

OCCURRENCE OF SPOILAGE ORGANISMS IN FRESH-CUT PRODUCTS

Deterioration of fresh-cut products occurs after packaging, during retailing, andalthough this may be due to the natural, physiological response of the plant tissue,microorganisms make a significant contribution to onset of spoilage and decreasein product shelf life (Lund, 1971, 1992; Nguyen-The and Carlin, 1994; Brackett,1997; Heard, 1999b). To be able to predict when and where spoilage will occur, weneed to know which organisms are present and which organisms are responsible foronset of spoilage. And, we need to understand some of the characteristics of theorganisms.

Relatively few studies have reported the occurrence of individual, includingspoilage, organisms in fresh-cut fruits and vegetables. Table 7.6 summarizes thepredominant microorganisms present in fresh-cut products, including salads that aremixed with mayonnaise and that may contain other ingredients such as cheese,seafood or meat. Although not strictly considered to be fresh-cuts, these are commonways of packaging and retailing minimally processed salad vegetables, and themicrobiological consequences of adding such ingredients should be considered.

Pseudomonads are the most common organisms isolated from fresh-cut vege-table salads. Biovars of Pseudomonas fluorescens are generally the main species(Denis and Picoche, 1986; Marchetti et al., 1992) and have been reported in mixedsalads as well as lettuce salads at populations of up to 107 cfu/g salad (Denis andPicoche, 1986; Geiges et al., 1990). Marchetti et al. (1992) found that P. fluorescensoccurred at higher frequency (23% of 162 isolates) than all other bacteria presenton both lettuce and mixed salads. Other species often present on leafy vegetablesinclude Pseudomonas putida, Pseudomonas chloraphis, Pseudomonas corrugata,Pseudomonas cepacia, Pseudomonas paucimobillis, Pseudomonas marginalis (P. fluo-rescens biotype II) and Pseudomonas viridflava (Table 7.6). Nguyen-The and Prunier(1989) observed that Ps. marginalis was the predominant organism on chicory-endiveleaves. They concluded that it was a weak plant pathogen only causing spoilage if

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TABLE 7.6Predominant Spoilage Microflora in Salads

Spoilage Organism Salad Ref.

PseudomonadsPseudomonas fluorescens Carrots, lettuce, mixed salad, chicory, potato salad,

dryslaw, tabouli1,2,3,4,5,6,9,11

Pseudomonas fragi Potato salad, dryslaw, tabouli 11Pseudomonas putida Carrots, lettuce, mixed salad, alfalfa sprouts, potato

salad, dryslaw, tabouli1,2,3,4,9,11

Pseudomonas marginalis Carrots, lettuce, mixed salad, chicory leaf, potato salad

1,3,9,11

Pseudomonas cepacia Carrots, mixed salads, chicory 5Pseudomonas chicorii Chicory 9Pseudomonas fulva Chicory 9Pseudomonas paucimobilis Carrots, mixed salad, chicory 5Methylobacterium mesophilica Carrots, mixed salad, chicory 5Pseudomonas viridiflava Carrots, lettuce, mixed salad 1Stenotophomonas maltophilia Carrots, lettuce, mixed salad, potato salad, dryslaw 1,11Pseudomonas chloroaphis Prepared salad 4Pseudomonas corrugata Chicory, sprouts, potato salad, dryslaw, tabouli 9,11Flavimonas oryzihabitans Tabouli 11

Other BacteriaAgrobacterium radiobacter Potato salad 11Acinetobacter spp. Tabouli 11

Coryneform bacteria Carrots, lettuce, mixed salad 1Flavobacterium sp. Carrots, lettuce, mixed salad 1Enterobacter agglomerans Mixed salad, chicory leaf, salad leaves, prepared

salad, dryslaw, tabouli2,3,4,6,7,11

Enterobacter amnigenus Tabouli 11Enterobacter gervoiae Dryslaw 11Erwinia carotovora Mixed salad 2,6Enterobacteriaceae** Chicory, sprouts 9Klebsiella terrigena Dryslaw 11Lactobacillus spp. Carrots, lettuce, mixed salad 1,6Leuconostoc spp. Carrots, lettuce, mixed salad, potato salad, dryslaw,

tabouli1,2,11

Rahnella aquatilis Potato salad, dryslaw 11Serratia marcescens Tabouli 11Yersinia intermediata Potato salad 11

YeastsCandida spp. Salad mix, mayonnaise-based salads,* carrots,

chicory5,6,8,10

Cryptococcus albidus Lettuce 6Cryptococcus laurentii Lettuce, carrots, mixed salad, chicory 5,6

(continued)

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present on leaves in excess of 108 cfu/g. Jayasekara (1999) enumerated pseudomonadsand related species on several salads, including tabouli, dryslaw mix and potatosalad. Most commonly isolated species included Ps. fluorescens, Ps. corrugata, Ps.putida and Ps. marginalis. Other commonly isolated organisms included species fromthe Enterobacteriacae and lactic acid bacteria. Coryneform bacteria and species ofLactobacillus and Leuconostoc are commonly isolated from whole vegetables andmay contribute to the spoilage of processed salads during storage (Brackett, 1994, 1997).Of the lactic acid bacteria, Leuconostoc are reported to predominate, in particular,Leusonostoc mesenteroides (Carlin et al., 1989). Populations of Lactobacilli of around104 cfu/g have been reported on fresh-cut pineapple and cantaloupe after processing,increasing to 107 cfu/g after 12 days of refrigerated storage (O’Conner-Shaw et al.,1994; Portella et al., 1997 cited in Lamikanra et al., 2000).

Bennick et al. (1998) reported the occurrence of various species of Enterobac-teriaceae, including Enterobacter cloacea, Pantoea agglomerans and Rahnella aqua-tilis on mung bean sprouts and chicory endive. Pseudomonas species such as Ps.fluorescens, Ps. corrugata and Ps. viridiflava also were detected on fresh and spoiling

TABLE 7.6Predominant Spoilage Microflora in Salads (Continued)

Spoilage Organism Salad Ref.Debaryomyces hansenii Salad mix, mayonnaise-based salads* 6,8Pichia fermentans Lettuce, salad mix 6Pichia membranifaciens Mayonnaise-based salads* 8,10Saccharomyces cerevisiae Salad mix, mayonnaise-based salads* 6,8Saccharomyces dairensis Mayonnaise-based salads* 8Saccharomyces exiguus Mayonnaise-based salads* 8Torulaspora delbrueckii Salad mix, mayonnaise-based salads* 8,10Trichosporon cutaneum Lettuce 6Yarrowia lipolytica Mayonnaise-based salads* 8Zygosaccharomyces bailii Mayonnaise-based salads* 8

MoldsAspergillus niger Lettuce, mayonnaise-based salads* 6,8Botrytis allii Lettuce 6Penicillium chrysogenum Mayonnaise-based salads* 8

References—1: Denis and Pioche (1986); 2: Brocklehurst et al. (1987); 3: Nguyen-The and Prunier (1989);4: Geiges et al. (1990); 5: Marchetti et al. (1992); 6: Magnuson et al. (1990); 7: Gras et al. (1994);8: Hunter et al. (1994); 9: Bennick et al. (1998); 10: Birzele et al. (1997); and 11: Jayasekara (1999).

* Mayonnaise-based salads include coleslaw, rice salads, potato salads, fruit and nut salads, prawn andpasta salads and other miscellaneous salads.

** Enterobacteriaceae isolates include Rahnella aquatilis, Serratia odorifera, Escherichia vulneris, Kleb-siella oxytoca, Enterobacter cloacae, Erwinia amlyovora, Enterobacter intermedius, Kluyvera cryocre-scens, Serratia proteamaculans, Buttiauxella agrestis and Enterobacter cloacae.

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vegetables. Predominant species reported on vegetables such as broccoli, endive andsprouts include the fluorescent pseudomonads and species of Klebsiella, Serratia,Flavobacterium, Xanthomonas, Chromobacterium and Alcaligenes (Table 7.6).

A number of yeast species have been associated with dry and dressed, low-pHsalads. Although yeasts have been implicated in the spoilage of fermented vegetableproducts, their role in the spoilage of fresh vegetables is not well studied. Spoilagedefects reported in the literature include soft rot formation in onions, surface defectsof vegetables such as bell peppers and discoloration of cabbages caused by Klyver-omyces spp. (Fleet, 1992). The main yeasts associated with vegetable spoilage areCryptococcus species, in particular, Cryptococcus laurentii and species of Candida.The more fermentative species, S. exiguus, Saccharomyces cerevisiae and S. dairen-sis occur mainly in salads dressed with mayonnaise. Spoilage defects caused byfermentative yeasts are off-odors, gassiness and growth on the surface of the salad(Denis and Buhagiar, 1980; Fleet, 1992). The origin of Saccharomyces species inprocessed salads is not clear but may be the result of contamination from theprocessing environment, as these species are not commonly found in unprocessedfruits and vegetables (Deak and Beuchat, 1987). Spoilage as a result of mold growthdoes not appear to be a major problem for ready-to-eat salads, although the problemis not well documented in the literature. A wide variety of fungi is commonly foundon the surface of other vegetables and fruits at the time of harvest (Lund andSnowden, 2000; Nguyen-The and Carlin, 2000). However, the high moisture contentof the processed fruits and vegetables and modified atmospheres in packaged saladsfavor proliferation of the faster-growing bacteria and yeast species, thus reducing orinhibiting the growth of molds (Nguyen-The and Carlin, 2000). Botrytis, Aspergillusand Penicillium spp. have been reported to occur on salad vegetables such as lettuce(Magnuson et al., 1990; Hunter et al., 1994). In a recent study of the quality of minimallyprocessed cantaloupe, Lamikanra et al. (2000) reported growth of Gram-negativeand Gram-positive bacteria with only minimal presence of mold.

CHARACTERISTICS OF SPOILAGE ORGANISMS

Our understanding of the microbial spoilage of fresh-cut products is incomplete,but we can draw on our knowledge of spoilage of whole fruits and vegetables andother foods to predict spoilage patterns and microbial interactions. Some character-istics of those organisms known to occur on fresh-cut products are presented in thissection.

Pseudomonads and Related Species

The family Pseudomonadaceae consists of the four genera: Pseudomonas, Xanth-omonas, Zoogloea and Frauteuria (Palleroni, 1992). The type genus is Pseudomonas,and members are often referred to as pseudomonads. They are Gram-negative rods,occurring singly or in pairs, motile with one or more polar flagella, strictly aerobiccatalase positive and oxidase positive or negative. Pseudomonads are also charac-terized by their ability to grow in simple media. In recent years, species of this genushave been characterized for taxonomic purposes by their RNA group (five RNA groups).

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Ps. aeruginosa (type species) and fluorescent species commonly associated withplants Ps. fluorescens, Ps. putida, Ps. chlororaphis, Ps. syringae and Ps. viridflavabelong to RNA group I. Pseudomonas fluorescens is a heterogeneous species thathas been subdivided into groups known as subspecies or biotypes/bivars A, B, C,D, E, F and G. They are grouped according to biochemical, physiological andnutritional characteristics (Stanier et al., 1966; Barrett et al., 1986). Pseudomonasfluorescens G is the most heterogeneous of all the biotypes and includes strains withirregular nutritional properties and characteristics. No methods for identification andsubdivision can entirely classify strains of this biotype (Palleroni, 1992, 1993). Theother major group of plant pathogens, the Pseudomonas cepacia-Pseudomonassolanacearum belongs to RNA group II.

The role of pseudomonads as spoilage organisms is well acknowledged in thefood industry. They are capable of synthesizing enzymes, even under refrigerationconditions that facilitate the breakdown of food components and cause spoilage(Cousin, 1982; Jayasekara, 1999). During the spoilage of fruits and vegetables,pseudomonads produce pectolytic enzymes to degrade the cell walls of the hosttissue. This results in maceration of the tissue. Other tissue-degrading enzymesproduced include cellulases, xylanases and glycoside hydrolases and lipoxygenase(Gross and Cody, 1985; Zhuang et al., 1994). Padaga et al. (1999) tested 165 bacterialisolates from broccoli florets for production of pectinolytic enzymes and lipolyticand proteolytic activity. Strains of Ps. fluorescens B were predominantly pectinolytic,producing pectate lyase, pectolytic and polygalacturonase activity. Proteolytic activ-ity and lipolytic activity were noted for pseudomonad isolates. Biosurfactants pro-duced by pseudomonads also assist in degradation of plant tissue. An example isviscosin, a potent peptolipid produced by a strain of Ps. fluorescens B. Viscosinproduction facilitates bacterial infection and spread of decay on unwounded broccoliflorets (Laycock et al., 1991). Padaga et al. (1999) reported that 50% of the strainsof Ps. fluorescens A, Ps. viridiflava, Ps. mendocina and Ps. fragii isolated frombroccoli were capable of producing biosurfactant. However, only 10% were strongproducers. Similar studies should be conducted for isolates from fresh-cut productsto determine the role of pseudomonads in onset of spoilage.

Pseudomonads may also contribute to the yellowing of vegetable products duringstorage, through the production of the ripening hormone ethylene. Weingart andVölksch (1997) and Weingart et al. (1999) studied the production of ethylene byPseudomonas. syringae. When inoculated into a weed, it was shown that enhancedethylene production during onset of disease was due to ethylene production by thebacteria. Padaga et al. (1999) also observed ethylene production by various strainsof Ps. fluorescens A and G from broccoli origin.

Lactic Acid Bacteria

The term lactic acid bacteria describes a number of genera of Gram-positive bacteria(rods and cocci) that are traditionally known as fermentative organisms associated withfermented food products and food spoilage. Those genera commonly associatedwith spoilage of foods include Lactobacillus, Leuconostoc and Pediococcus. Basedon chemotaxonomic and phylogenetic studies, these three genera are closely related,

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with overlap between them. There are three main groups: the Lactobacillus del-brueckii group, which includes mainly homofermentative lactobacilli; the Lactoba-cillus casei/Pediococcus group; and the Leuconostoc group, including some obligateheterofermentative lactobacilli (Stiles and Holzapfel, 1997). The habitats of speciesof the genera Lactobacillus and Leuconostoc include plants and plant material, soil,water and sewage and fruit and grain mashes (Stiles and Holzapfel, 1997). Leu-conostoc spp. may be present on growing, undamaged plants in relatively lownumbers, but numbers increase during maturation and later during harvest (Daeschelet al., 1987). Although considered desirable during the production of fermentedfoods, fermentation of sugars to produce acid and gas is undesirable in fresh-cutproducts. Their fermentative metabolism and ability to grow in anaerobic conditionsenables lactic acid bacteria to cause spoilage, such as souring of the product, gasproduction and slime formation (Carlin et al., 1990; Stiles and Holzapfel, 1997).

Enterobacteriaceae

The family Enterobacteriaceae consists of a number of genera of Gram-negativerod-shaped bacteria. The different genera with a variety of ecological niches, includ-ing plants, insects, animals and humans, may contaminate fresh-cut products on thefarm and during processing. The pathogenic species of this group are usually enu-merated using selective isolation media, but the nonpathogens are generally isolatedas a total group termed “coliforms.” With the exception of Erw. caratovora, a knownpostharvest pathogen (Nguyen-The and Carlin, 2000), reports of the occurrence ofindividual species in foods are rare. One such report by Bennick et al. (1998)described the presence of several species on fresh vegetables. They included Ent.cloacae, Pant. agglomerans, Rah. aquatilis, Erw. caratovora, Erw. amylovora, Kleb.oxytoca and Serratia. oderifera. The main characteristics of these organisms allowinggrowth and potential spoilage of fresh-cut vegetables are as follows:

• their ability to grow as facultative anaerobes, thus they can survive in themodified atmosphere of packaged salads (Bennick et al., 1998)

• their ability to ferment glucose to produce acids, alcohols and esters (Adamsand Moss, 1995)

The role of these organisms in the spoilage of fresh-cut products is not wellunderstood and is an area for future research.

Coryneform Bacteria

The term “coryneform bacteria” is a general term used for practical purposes todescribe a large, diverse group of bacterial taxa that are Gram positive, nonsporing,irregular-shaped rods. They are mostly aerobic, and many of the genera are pigmented.Some of the genera, including Arthrobacter, Rhodococcus and Brevibacterium mayhave a distinct rod-coccus growth cycle, while others do not display such obviousirregularity (Coyle and Lipsky, 1990). The coryneform groups are well distributedin nature and are found in soil, on plants and in food processing environments.

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222 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Taxonomic classification of the group is confusing, and identification is difficult,because characteristics used in conventional identification of bacteria such as mor-phology and physiology are of little value to separate the taxa. The main approachesto identification of organisms within the corynefom group are the use of chemotax-onomic methods, DND-DNA homology and 16sRNA cataloging (Minnikin andGoodfellow, 1980; Gobbetti and Smacchi, 2000). However, little is known about theoccurrence and activity of individual genera or species of coryneform bacteria infoods. Currently, the group consists of the following genera—Arthrobacter, Brevi-bacterium, Rhodococcus, Curtobacterium, Micobacterium, Aureobacterium, Coryne-bacterium, Agromyces, Cellulomonas and Oerskovia. One genus known to occur invegetable products is Arthrobacter. Some of the characteristics of this species willbe discussed.

Arthrobacter Arthrobacter spp. have a rod-coccus growth cycle, as mentioned previously, do notform endospores and may be either motile or nonmotile by one subpolar or fewlateral flagella. They have a respiratory metabolism and are never fermentative. Theyoften occur in soil and in the rhizospheres of plants, exhibiting extreme resistanceto dry conditions and periods of starvation. Isolates from soils often have the abilityto degrade polymeric compounds, although they do not often produce pectolyticenzymes (Gobbetti and Smacchi, 2000). Arthrobacter spp. are recognized as playinga role in the ripening of smear cheeses, contributing to aroma, flavor and texturedevelopment. These changes are facilitated by the production of lipolytic and proteolyticenzymes. There is less information available describing the activity of Arthrobacterspp. on vegetables. Gobbetti and Smacchi (2000), reviewing the role of Arthobacterspp. in foods, report that they are chitinolytic bacteria, able to degrade fungal hyphae,thus destroying some soilborne fungal pathogens. They also report that Arthrobacterspp. have been known to degrade pesticides in polluted and cold environments.Arthrobacter spp. isolated from broccoli florets were reported by Padaga et al. (1999)to produce biosurfactants and tissue-degrading enzymes including pectolytic, lipolyticand proteolytic enzymes. These species also produced polyphenol oxidase activityand were capable of forming unpleasant aroma compounds such as methanethiol.Further investigation is necessary to determine the significance of coryneform bacteriasuch as Arthrobacter spp. in the fresh-cut environment.

Yeasts and Molds

A wide variety of species of molds are known to cause postharvest disease in fruitsand vegetables. However, in the fresh-cut environment, faster-growing yeasts tendto outgrow molds to cause spoilage. Characteristics of yeasts will be discussed.Yeasts are single-celled eukaryotic organisms of which many genera are associatedwith the fermentation and spoilage of foods. Fermentative species of yeasts such asKloeckera and Hanseniaspora occur naturally on the surfaces of fruits and arecapable of causing fermentative spoilage (Barnett et al., 2000). Other fermentativespecies such as S. cerevisiae and S. exiguus may contaminate fruits during processingand cause explosive fermentative spoilage. Growth and fermentation do not usually

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Microbiology of Fresh-cut Produce 223

occur until the fruit is damaged, allowing leakage of juices and sugars. Thus, in thefresh-cut environment (and in low-pH dressed salads), yeast growth and spoilage offruits is predictable. The characteristics of yeasts that allow growth are their abilityto ferment simple carbohydrates to produce alcohol, gas and flavor components,such as esters, acids and higher alcohols, and the ability of some species to grow atrelatively low temperatures (10–15°C). Less fermentative species, such as P. mem-branifaciens, Candida krusei and Kluyveromyces, may also spoil fresh-cut productsthrough the formation of films or off-odors (Fleet and Heard, 1992; Fleet, 1993;Heard 1999a).

FOODBORNE PATHOGENS

THE ASSOCIATION OF FOODBORNE PATHOGENS WITH FRESH-CUT SALADS

Fresh-cut products have been linked with outbreaks of foodborne disease, and food-borne pathogens may form a part of the microflora of these products. Historically,epidemiological surveillance of foods for the presence of pathogens has concen-trated on foods of animal origin. Although fruits and vegetables have long beenknown as sources of infectious microbial agents, there is very little evidence docu-menting the risks to public health. Increased consumption of produce and the growthof the fresh-cut industry have prompted interest to investigate the association ofpathogens with these products. This section will briefly describe the pathogens ofconcern, the diseases they cause, their origin and some of the characteristics thatenable them to survive in fresh-cuts and the processing environment. Currentapproaches to detection of pathogens will be mentioned, and gaps in knowledge willbe highlighted.

In recent years, a number of review papers have discussed the occurrence offoodborne pathogens in fresh produce and fresh-cut products (Fain, 1996; Beuchat,1996, 1998; Francis et al., 1999). These reports have highlighted the gaps in knowl-edge regarding the organisms of most concern and emphasized the need for full riskanalysis of fresh-cut production from the farm to the consumer (Beuchat, 1998). Itis generally accepted that the pathogens of concern in fresh-cut salads are similarto those present on raw vegetables (Nguyen-The and Carlin, 2000). Due to theworldwide concern over listeriosis, many studies have focused on the incidence ofL. monocytogenes in processed, refrigerated vegetable products (Nguyen-The andCarlin, 2000). However, other bacterial pathogens reported to be of concern are Cl.botulinum, enterohemorraghic E. coli including serotypes O157:H7 and O111, Shigellaspp., B. cereus, Aeromonas hydrophila and Yersinia enterocolitica (Fain, 1996).Pathogens also associated with raw vegetables include Vibrio chloerae (isolated fromcabbage), St. aureus and Salmonella spp. (isolated from salad vegetables) andCampylobacter jejuni (found in retail mushrooms) (Doyle and Schoeni, 1986; Satchellet al., 1990; Fain, 1996; Francis et al., 1999). Some examples of foodborne pathogensisolated from fresh-cut salad products are listed in Table 7.7. Table 7.8 lists a numberof outbreaks of foodborne disease associated with fresh-cuts. Nonbacterial patho-gens may also be transmitted by fresh produce, originating from wash water and being

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224 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

transmitted from the food to food handlers. These include viruses, such as the Norwalkvirus and hepatitis A, and the parasites Giardia and Cryptosporidium. Characteristicsof selected pathogens of concern will now be discussed in more detail.

Pathogens of Concern

Listeria MonocytogenesListeria monocytogenes is a Gram-positive bacteria capable of causing foodbornedisease in humans. The infective dose of this organism is not yet defined, althoughit is currently thought that a dose of greater than 103 cfu/g is necessary to causedisease. Illness usually occurs in those who are immunosuppressed, such as inpregnant women, neonates, cancer patients and the elderly, and although manypeople may be without symptoms, the clinical manifestations of the disease includemild febrile gastroenteritis, conjunctivitis, meningitis, septicemia and spontaneousabortion and death (Sutherland and Porritt, 1997; Farber and Peterkin, 2000).

Although the risk of listeriosis is considered minor by many authors, it is theseverity of the disease that causes concern. Foods identified as high risk includerefrigerated, minimally processed products such as fresh-cut salads (Sutherland andPorritt, 1997). Listeria monocytogenes can survive and grow at both ambient andrefrigeration temperatures, and it is facultatively anaerobic, enabling it to persist in

TABLE 7.7Some Examples of the Occurrence of Foodborne Pathogens in Fresh-cut Salad Products

Pathogenic Species Product (++++ Origin) Occurrence Ref.Aeromonas hydrophila Prepared salads (U.K.) 21.6% Fricker and

Tompsett (1989)Campylobacter Vegetable salads

(Germany)0% (20 samples) Karib and Seeger

(1994)Cryptosporidium oocysts

Lettuce (Costa Rica) 2.5% (2/80 samples) Monge and Chinchilla (1996)

E. coli O157:H7 Cabbage (Mexico) 25% Beuchat (1996)Listeria monocytogenes Coleslaw (Canada) 2.2% (2/92 samples) Schlech et al. (1983)Listeria monocytogenes Prepacked salad

(N. Ireland)14.3% Harvey and Gilmour

(1993)Listeria monocytogenes Coleslaw (U.K.) 7.7% (3/39 samples) MacGowan et al.

(1994)Listeria monocytogenes Prepared vegetables

(U.K.)3.8% (1/26 samples) MacGowan et al.

(1994)Salmonella Mixed salad vegetables

for retail use (U.S.)0% (0/63) Lin et al. (1996)

Shigella Salad vegetables (Egypt) 1.2% Satchell et al. (1990)Staphylococcus spp. Salad vegetables (Egypt) 8.3% Satchell et al. (1990)Yersinia enterocolitica Packaged vegetable

products (France)22–56% (100 samples)

Manzano et al. (1995)

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Microbiology of Fresh-cut Produce 225

these products. The organism may enter the processing chain from the farm or fromthe processing environment. It is present in the intestinal tract of animals and humans,and it is found to be widespread in soil and in sewage. It is also a saprophyte andcan survive on decaying plant material (Beuchat, 1998), and it is disseminated onfarms by animals grazing on decaying plants, spreading their feces onto fresh fields.If it contaminates the processing environment, it may colonize processing surfaces,surviving in drains, cracks in floors and walls and in crevices in equipment. Thereare reports describing survival of the organism and formation of biofilms on surfacesin food-processing environments, particularly in drains. It may also be transmittedby aerosols and on workers’ hands (Sutherland and Porritt, 1997; Beuchat, 1998).

TABLE 7.8Examples of Outbreaks of Foodborne Disease Associated with Consumption of Fresh-cut Products

Pathogenic Species

Product(++++ Origin)

Source of Contamination

Number of Cases of Disease Ref.

Cyclospora cayetanensis

Raspberries (U.S., Canada—produce imported from Guatemala)

Spraying with fungicides prepared with contaminated water

>1000 Hertwaldt et al. (1997), Tauxe et al. (1997)

Escherichia coli O157:H7

Mixed lettuce (U.S.) Bad manufacturing practice, possibly fecal contamination

26 people De Roever (1998)

Hepatitis A virus Diced tomatoes Food handler 92 Williams et al. (1994) cited by Lund and Snowden (2000)

Norwalk virus Fresh-cut fruit Food handler >217 Herwaldt et al. (1997)

Listeria monocytogenes

Coleslaw (Canada) Sheep manure 34 cases Schlech et al. (1983)

Salmonella spp. Precut watermelons (U.S.)

Stored without refrigeration

18 cases Blostein (1993), CDC (1979, 1991)

Cantaloupe (Mexico and Central Amercia)

Not known, obtained from salad bars

>245 (estimated 25,000)

CDC (1991), Tamplin (1997)

Shigella sonnei Shredded lettuce Processing 347 people Davis et al. (1988)Shigella spp. Fruit salad (U.K.) Not known 36 O’Brien (1998)Vibrio cholerae Cabbage (Peru) Organic

fertilizer of polluted water

— Swerdlow et al. (1992)

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226 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Listeria monocytogenes has been isolated from prepackaged mixed vegetableproducts, chicory endive and lettuce fresh-cuts, sliced cucumber and fruits, such astomatoes and cantaloupe, and has also been implicated in foodborne disease out-breaks in several countries (Beuchat and Brackett 1991, Beuchat 1998). For example,vegetable mix for coleslaw was implicated as the vehicle for L. monocytogenes,causing an outbreak of listeriosis in Canada in 1981. However, most reports oflisteriosis associated with fresh produce are linked to the consumption of wholefruits and vegetables. Further investigation is required to quantify the risks of list-eriosis associated with fresh-cut products.

Enteric Pathogens (Family Enterobacteriaceae)

Escherichia ColiEscherichia coli is part of the natural microflora of the intestinal tract of warm-bloodedanimals and humans. However, there are also strains capable of causing gastrointestinaldisease in humans. These strains are grouped as the enterotoxigenic, enterohemor-rhagic, enteropathogenic and enteroinvasive strains of E. coli (Doyle et al., 1997).Enterotoxigenic E. coli causes traveller’s diarrhea, and contaminated produce maywell be a source of this organism (Beuchat, 1998). Similarly, enteropathogenicE. coli causes gastroenteritis symptoms in adults and infants, and enteroinvasive E. coliinvades the colonic epithelial tissue lining the colon, resulting in the onset of bloodydiarrhea (Doyle and Padhye, 1989; Desmarchelier and Grau, 1997). The final group,enterohemorrhagic E. coli is probably the most dangerous to humans. The infectiousdose of this organism has been shown to be as low as two cells in 25 g of food, andit is now believed that the infectious dose is less than 100 cells/g food (Willshawet al., 1994; Griffin et al., 1994; Doyle et al., 1997). Although the pathogenicity is notfully understood, it produces a number of verotoxins (cytotoxins to the African greenmonkey kidney cells), depending on the strain and enterohemolysin (Desmarchelierand Grau, 1997). The most severe signs are seen in the elderly and in children. Itcauses hemorrhagic colitis, hemolytic uremic syndrome (usually in children) andthrombocytopenia purpura (in adults). Deaths have been reported mainly in theelderly (Doyle et al., 1997).

Fresh produce can become contaminated with any one of these organisms in thefield, through contact with contaminated animal droppings, particularly from rumi-nants, or from organic fertilizers, such as uncomposted manure. Other potentialcontamination sources are the water used, workers’ hands and wind and dust con-tamination, as described in a previous section (pages 190–197). Survival of theorganism and mechanisms of contamination in the processing environment have notbeen studied. Enterohemorrhagic E. coli O157:H7 has been recognized in recentyears as a food-related pathogen and has been responsible for outbreaks linked toa wide range of foods, including fresh produce. This organism may grow on pro-cessed fruits such as watermelon and cantaloupe (del Rosario and Beuchat, 1995),shredded lettuce, sliced cucumbers and sprouts (Abdul-Raouf et al., 1993; Zhao et al.,1993; Diaz and Hotchkiss et al., 1996; Nathan, 1997). The organism can grow attemperatures down to around 7–8°C and has been shown to survive under acidic

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Microbiology of Fresh-cut Produce 227

conditions, in apple juice, at this temperature for up to 12 days (Zhao et al., 1993).Fluctuations in handling and storage temperatures of fresh-cut products, including cutfruits such as cantaloupe and other melons, may provide opportunity for this organismto survive, creating a public health risk. It is of some concern that most research onsurvival, detection and enumeration of Enterohemorrhagic E. coli has focused onjust one serotype, O157:H7. Other serotypes, including O111:NM, O26:H11 andO26:HNM are also frequently implicated in outbreaks of disease (Desmarchelierand Grau, 1997), yet there is little evidence to document their incidence in foods.This is an area for future study.

ShigellaThe genus Shigella is closely related to E. coli (ICMSF, 1996). The genus is dividedinto four species: Shigella dysenteriae, Shigella sonnei, Shigella flexneri and Shigellaboydii, all of which can cause shigellosis or bacillary dysentery in humans. Invasiveserovars of Sh. dysenteriae produce a cytotoxin called a Shiga toxin. Noninvasiveserovars and other Shigella species produce only low levels of cytotoxicity and showendotoxic and neurotoxic activity (Lightfoot, 1997; Lampel et al., 2000). Outbreaksof shigellosis are generally linked to water of food contaminated with human feces.Thus, fresh produce can become contaminated through the use of contaminatedirrigation water, the use of raw sewage as fertilizers, insect transfer or human contact(Beuchat, 1998). Shigella species can survive on shredded lettuce under refrigerationfor up to three days without populations decreasing and can also survive on slicedfruits, including watermelon and raw papaya (Escartin et al., 1989; Satchell et al.,1990). Processed fruits and vegetables have been implicated in a number of outbreaksof shigellosis. Salad vegetables, cantaloupe and potato salad are examples of theassociated products (Formal et al., 1965; Frelund et al., 1987; Dunn et al., 1995).

Salmonella Within the genus Salmonella, differentiation into species is based on antigenicdifferences. There are currently over 2370 serovars recognized, however, only 200are known to cause disease in humans, including Salmonella typhi, the causativeagent in the disease typhoid (Jay et al., 1997; Beuchat, 1998; D’Aoust, 2000). Food-borne disease caused by nontyphoid serovars of Salmonella includes gastroenteritisand enterocolitis, with symptoms appearing from 8–72 h after food consumption.More severe complications include septicemia and onset of reactive arthritis (Jay et al.,1997). Salmonellae have been isolated from fresh produce, and fruits and vegetableshave been linked to outbreaks of salmonellosis (Hedburg and Olsterholm, 1993;Beuchat, 1998). Fresh produce may become contaminated with salmonellae eitherfrom sewage and contaminated water or from handling by infected workers.Although there are no reported cases of salmonellosis from fresh-cut products,Salmonella can grow on the surface of alfalfa sprouts (Jaquette et al., 1996), andthese are sometimes used as ingredients in mixed packaged products. Salmonellaedo not grow in foods at less than 7°C and, therefore, should not pose a risk to publichealth in fresh-cut products, provided they are maintained at refrigeration tempera-tures. Further investigation of the factors influencing survival of this pathogen inthe salad environment are recommended.

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228 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

SPORE-FORMING BACTERIA

Spore-forming pathogens such as Clos. perfringens, Clos. botulinum and B. cereusmay be isolated from soil and have been isolated from fresh and minimally processedvegetables and raw seed vegetable sprouts (Beuchat, 1996). Clostridium botulinumproduces potent neurotoxins that produce a range of symptoms in humans, includingnausea, diarrhea and vomiting and neurological symptoms such as blurred vision,dilated pupils, paralysis of motor nerves, loss of mouth and throat normal functions,lack of muscle coordination and other complications and possible death. Modifiedatmosphere conditions favor the development of the organism, and although toxinproduction has not been detected in vegetables stored at refrigeration temperatures(Beuchat, 1996), care should be taken to avoid temperature increases, to preventgermination of spores, to prevent growth of vegetative cells and to prevent toxinproduction. Clostridium perfringens and B. cereus produce enterotoxins responsiblefor causing abdominal cramps and diarrhea (Bates, 1997; Jensen and Moir, 1997;Labbé, 2000; Lund and Peck, 2000). There is also an emetic strain of B. cereus thatcauses rapid onset of disease characterized by acute nausea and vomiting but usuallyno diarrhea. Clostridium perfringens cells die at temperatures below 10°C but cangrow at 15°C. The risk to public health arises if products contaminated with theseorganisms are handled in such a way as to enable spore germination and outgrowthof the vegetative cell, for example, when temperature fluctuations occur duringhandling, transport and retailing of the finished product. Psychrotrophic strains ofB. cereus have been isolated from foods (Jensen and Moir, 1997) and may pose arisk to public health if present in refrigerated fresh-cut products.

Staphylococcus AureusStaphylococcus aureus (Gram-positive cocci) has been isolated from vegetables andfresh-cut products (Abdelnoor et al., 1983; Houang et al., 1991), but there have beenno reports of staphylococcal food poisoning from such products. This organism isthought to originate from handling by workers. It is often present in the nasalpassages and on the hands of humans. It is generally accepted that Staph. aureusdoes not compete well in fresh foods, where there is a diverse microflora (Baird-Parker, 2000). However, there have been no reports of the factors influencing thegrowth of the organisms in the fresh-cut environment.

Campylobacter Campylobacter jejuni (Gram-positive spiral rods) is found in the intestinal tract ofa wide variety of wild and domestic animals. It is a common cause of bacterialenteritis in many countries and is generally associated with food poisoning outbreaksinvolving animal products. Symptoms of the disease include acute diarrhea lastingfor up to five days accompanied by fever and abdominal pain. There are someincidences of infection arising from contamination of fruits and vegetables (Beanand Griffin, 1990; Castillo and Escartin, 1994; Harris et al., 1989) presumablycontaminated from animal waste. Cross-contamination between animal and vegeta-ble products may occur where non-vegetable ingredients are added to salads.Although the optimum growth temperature is 42°C, it grows under microaerophilicconditions similar to those of packaged fresh-cuts.

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Microbiology of Fresh-cut Produce 229

Yersinia EnterocoliticaYersinia spp. are Gram-negative rods or coccobacilli. Yersinia enterocolitica is partof the natural intestinal flora of swine, although it is also a psychrotroph that cangrow at temperatures as low as 0°C (Barton et al., 1997). This pathogen is recognizedto produce a wide range of clinical and immunological symptoms. The most commonis enterocolitis, seen mainly in young children, and pseudoappendicitis, occurringin older children and adolescents (Barton et al., 1997). It may contaminate freshproduce from feces and through cross-contamination in processing establishments.Yersinia spp. have been isolated from fresh fruits and vegetables, including saladvegetables such as lettuce and grated carrots, although it has not been implicated indisease outbreaks from eating produce (Beuchat, 1996; Nesbakken, 2000). They posea potential risk to public health in fresh-cut products because of their ability to growat low temperatures. An investigation of their incidence in fresh-cuts is warranted.

Aeromonas SpeciesAeromonas are Gram-negative rod-shaped coccoid bacteria that are ubiquitous tomost aquatic environments and occur in a wide variety of foods (Palumbo et al.,2000). Not all strains are pathogenic, and aeromonads are often responsible forspoilage of foods. Aeromonads may contaminate fresh fruits and vegetables fromwash water and possibly through cross-contamination from seafood, meat or poultry.Some strains can grow at refrigeration temperatures, reaching up to 106 cfu/g onvegetables such as asparagus, broccoli and cauliflower after two weeks of storage at4°C (Berrang et al., 1989). They can also survive under modified atmosphere condi-tions. The group most affected by pathogenic Aeromonas are the young. The organismcauses a self-limiting illness characterized by diarrhea and mild fever. There are alsoreports of cholera-like symptoms (Kirov, 1997).

Vibrio SpeciesVibrio species (Gram-negative vibrio-shaped rods) occur predominantly in estuarinewaters, and foodborne disease from these organisms are usually associated with fishand seafood. Of the 12 pathogenic species, Vibrio cholera causes the most severedisease, cholera (Kaysner, 2000). Vibrio parahemolyticus is often associated withdisease outbreaks from under-cooked seafood. Onset of disease may be up to 96hours after food consumption and is characterized by symptoms including diarrhea,nausea, vomiting, abdominal cramps and fever (ICMSF, 1996). The potential riskof disease from this organism in association with fresh-cut products is from cross-contamination during handling and mixing in retail establishments.

VirusesViruses can be responsible for outbreaks of foodborne disease. They are excretedby infected individuals, and although they do not grow on food, they can survive inwater and sewage and may subsequently contaminate food such as fruits and veg-etables. Over 150 types of enteric viruses representing four viral families can bepresent in raw sewage, and they cause a range of diseases including respiratoryinfections, skin disorders, meningitis and gastroenteritis (Grohmann, 1997; OwenCaul, 2000). Diseases caused by hepatitis A, rotavirus and Norwalk–like viruses(small, round-structured viruses) have most commonly been linked to fresh produce,

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230 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

the vehicles included lettuce, chopped tomatoes and strawberries (Beuchat, 1998).Other viruses linked to foodborne disease are astroviruses, enteroviruses, parvovi-ruses and adenoviruses. The factors affecting the survival of viruses on fruits andvegetables are not known and should be studied in the future.

ParasitesParasites are defined as eukaryotic organisms and may be classified in two maingroups, protozoa and helminths. Parasites are dependent on host organisms for sur-vival, and although their life cycles vary, they must all pass through an animal orhuman host to survive and reproduce (Goldsmid and Speare, 1997). They may infectfood from contaminated water or sewage, from food handlers or insects, or the parasitemay be ingested by animals and be present in animal flesh at the time of slaughter.Many parasites are worldwide in their distribution and are prevalent especially inThird World countries where sanitation and hygiene conditions are poor (Goldsmidand Speare, 1997). Protozoa most commonly associated with human infections areGiardia, Cryptosporidium, Cyclospora, Entamoeba, Toxoplasma, Sarocystis and Iso-pora (Goldsmid and Speare, 1997; Beuchat, 1998; Taylor, 2000). All of these parasitescause diarrhea-like symptoms except Toxoplasma which causes fetal damage andglandular fever-like syndrome (Goldsmid and Speare, 1997). A number of helminthshave also been associated with foods, including liver and intestinal flukes, particularly,the Trematoda. The epidemiology of these protozoa is not well understood andrequires more detailed surveillance. Protozoa such as Giardia lamblia and Cyclosporacayetanensis have been linked with foodborne disease, where the food vehicle wasfresh produce (Beuchat, 1998). Cryptosporidium has been found on a range ofvegetables, including lettuce, cucumbers, carrots, and tomatoes (Monge and Chinchilla,1996). The factors affecting the survival of parasites in processed fruits and vegetablesare not well understood and warrant investigation.

DETECTION OF PATHOGENS IN FRESH-CUT PRODUCTS

Surveillance programs in a number of countries have investigated the incidenceof pathogens in fresh-cuts and the involvement of fresh-cut salads in outbreaks offoodborne disease. Overall, the incidence of pathogens in these products is low, andpathogens such as Salmonella, often found on whole fruits and vegetables, have notbeen isolated from processed products. Nguyen-The and Carlin (2000) attribute thelow incidence of pathogens in these products to improved processing techniquesand the implementation of quality assurance or good manufacturing practices. How-ever, because there are reported outbreaks of foodborne disease associated withfresh-cuts, and because we have not yet fully investigated the microbial ecology ofthese products, we cannot assume that pathogens are not present. This is especiallyimportant in countries where there is no legislation enforcing the use of qualityassurance programs or prohibiting the use of contaminated water for irrigation ofcrops (Beuchat, 1998). The adequacy of methods for the detection of foodbornepathogens in the fresh-cut salad environment should be investigated, and wherenecessary, improved methods should be sought after to increase our ability to targetand detect pathogens. These issues must be addressed if we are to fully assess therisk to human health from the consumption of salads. Current knowledge of the

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Microbiology of Fresh-cut Produce 231

pathogens associated with fresh-cut products is incomplete and largely qualitative.Questions to be answered include the following:

1. Does the background flora of these products influence the survival ofpathogens?

2. Does the background flora interfere with the detection of pathogens inthese products?

3. Can pathogens be detected in the fresh-cut environment using currentdetection methods?

Methods are also needed to facilitate monitoring of viruses and parasites infresh-cut products to determine the extent of the risk to public health.

Pathogens are often present in foods in low numbers, or they may be trapped ina biofilm or in pores of the food and are subsequently difficult to detect. To facilitatedetection, enrichment techniques, requiring up to four to five days to complete, areused to allow growth of the pathogen within the food. Such techniques are used todetect organisms such as L. monocytogenes, Shigella, Salmonella, E. coli O157:H7,C. jejuni and Yersinia (Barton et al., 1997; Desmarchelier and Grau, 1997; Jay et al.,1997; Lightfoot, 1997; Sutherland and Porritt, 1997; Wallace, 1997). However, selectiveenrichment techniques for isolation of pathogens from foods provide only qualitativeinformation about the presence or absence of the organisms. Also of concern, manycountries use standardized methods for detection of pathogens in foods that havenot been tested for validity or reliability for use in the salad environment. Two recentstudies (unpublished work) showed that the microbial ecology of vegetable productsis so complex that conventional methods alone cannot sufficiently reveal the diversityof the microbial species present. These studies revealed that when fresh-cut saladswere inoculated with either L. monocytogenes or E. coli O157:H7 at 1–10 cells/gand 10–100 cells/g, detection of these pathogens using the appropriate standard methodwas obscured by the indigenous flora.

Future considerations for microbiological methods for detection of pathogensin fresh-cut salads are as follows:

1. Validation of methods for detection of pathogens in fresh-cut salads2. Reduction of time for sampling and detection (may include adoption of

rapid methods) 3. Development of quantitative sampling and detection methods to enable

study of the complete microbial ecology of these products

BIOCONTROL

There are very few hurdles in packaged fresh-cut products to prevent the growth ofmicroorganisms. The products are washed to remove excessive contamination, butafter processing, the main controls used are storage at refrigeration temperature andpackaging in modified atmospheres. Current approaches to food preservation advocatethe hurdle concept, in which multiple factors are used to prevent microbial growth

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(Leistner, 1995). Unfortunately the “fresh” nature of minimally processed fruits andvegetables prevents the use of traditional processing such as cooking/heating, andconsumers are demanding that our foods contain no chemical preservatives. Thus,application of biocontrol concepts may be useful to create extra preservation hurdlesfor fresh-cut products. Biocontrol methods include the use of the following:

1. Antagonistic organisms to control growth of either spoilage or pathogenicspecies (biopreservation)

2. Natural antimicrobial compounds to control microbial growth 3. Natural plant defenses to reduce microbial attack-induced resistance

BIOPRESERVATION

The influence of naturally occurring microorganisms of vegetables on pathogenicbacteria has been described in the literature. For example, Carlin et al. (1996) reportedthat the indigenous microflora isolated from endive leaves could competitively inhibitL. monocytogenes. Similarly, Babic et al. (1997) reported that freeze-dried spinachpowder, containing a mixture of mesophilic aerobic microorganisms, was inhibitoryto L. monocytogenes. Francis and O’Beirne (1998) found that mixed populations ofbacteria isolated from shredded lettuce generally diminished the growth of Listeriainnocua, when mixed in model media, and they concluded that species of Enterobactercompeted with the Listeria species. These authors suggested that the natural back-ground flora could be an important influence on growth of Listeria species on lettuce.

Pseudomonas spp. have been used as antagonists for postharvest applications.Species such as Ps. chlororaphis, Ps. fluorescens, Ps. putida and Ps. syringae havealso been tested for siderophore production and antimicrobial activity against food-borne pathogens (Freedman et al., 1989; Laine et al., 1996; Janisiewicz et al., 1999).Almost all fluorescent pseudomonads and some nonfluorescent species producesiderophores, and these compounds are considered to be the main factors influencingthe biocontrol potential of pseudomonads (Jayasekara, 1999). Pseudomonads alsoproduce antifungal compounds [chitinase and laminarinase enzymes (Lim et al.,1991), syringomycin (Vassilev et al., 1996) and antimicrobial pigments (Dakhamaet al., 1993)] that may be applied to foods such as fresh-cut fruits. We may potentiallybe able to use selected spoilage flora of fresh-cut products to our advantage, throughexploitation of biocontrol characteristics. This will only be possible after completeinvestigation of the microbial ecology of the system.

Microorganisms such as lactic acid bacteria are used as biopreservative agentsin foods to inhibit the growth of other undesirable species. Mechanisms of antago-nism include competition for nutrients, binding of nutrients and production of met-abolic products with antimicrobial activity. Fermentation with lactic acid bacteria(LAB) is a traditional biopreservation method employed to increase the safety andquality of foods, including fruits and vegetables. Examples include fermented olives,sauerkraut and pickles (Stiles, 1996). In recent years, lactic acid bacteria have alsobeen used as competitive biocontrol agents and antagonists in nonfermented foods(Breidt and Fleming, 1997). These organisms are often present on the surface offruits and vegetables and, if encouraged, may reduce the growth of other indigenous

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Microbiology of Fresh-cut Produce 233

spoilage organisms or foodborne pathogens. Lactic acid bacteria are known to produceantimicrobial metabolites such as lactic and acetic acids, hydrogen peroxide andenzymes including lysozyme. Some strains of lactic acid bacteria also producebacteriocins (Holzapfel et al., 1995; Breidt and Fleming, 1997). These are describedas groups of potent antimicrobial peptides or proteins that are active against othermicroorganisms (Holzapfel et al., 1995). Several studies have described the use ofbacteriocin-producing lactic acid bacteria to improve the safety of ready-to-eat salads.Vescovo et al. (1995) investigated the effect of lactic acid bacteria on the mesophilicmicroflora during refrigerated storage of ready-to-eat vegetables. Strains of lacticacid bacteria selected for their ability to grow and produce antimicrobial compoundsat refrigeration temperatures inhibited the mesophilic flora, including enterococciand other coliforms, within three days of storage. In a subsequent study, Vescovo et al.(1996) applied antimicrobial-producing lactic acid bacteria to ready-to-eat vegeta-bles to prevent the growth of pathogens. Psychrotrophic strains of Lactobacillus casei,Lactobacillus plantarum and Pediococcus spp. inhibited A. hydrophila, L. monocy-togenes, Salmonella typhimurium and S. aureus in a range of vegetable salads. Furtherinvestigation with one strain, L. casei IMPC LC34, revealed that addition of thisstrain to ready-to-use vegetables resulted in reduction of total mesophilic counts andcoliforms counts and the disappearance of the pathogens A. hydrophila, Sal. typh-imurium and Staph. aureus after six days of storage. A culture permeate of this strainalso reduced counts of total flora, coliforms, enterococci and A. hydrophila in ready-to-eat vegetables (Torriani et al., 1997).

USE OF NATURAL ANTIMICROBIAL COMPOUNDS

Bacteriocins

Purified antimicrobial compounds such as bacteriocins may be added to fresh-cutvegetables to achieve a protective effect as an alternative to adding live cultures.Choi and Beuchat (1994) used a bacteriocin from Pediococcus acidilactici M toinhibit the growth of L. monocytogenes during kimchi fermentation. Addition ofcrude bacteriocin powder (10 mg bacteriocin/150 g salad) was initially lethal to L.monocytogenes and controlled growth of the pathogen for the duration of fermen-tation (16 days). A bacteriocin, Plantaricin D, has been isolated from Lactobacillusplantarum BFE 905, found in Waldorf salad (Franz et al., 1998). Plantaricin D wasfound to exhibit antilisterial activity, and the authors suggested use of the compoundor the biopreservative culture to improve the safety of ready-to-eat vegetables. Thecommercially available bacteriocin, nisin (from Lactobacillus lactis), has been usedin foods such as pasteurized cheese spreads to inhibit outgrowth of Clostridia spores,and it is approved in a number of European countries for addition to fresh cheese,processed vegetables and canned foods (Holzapfel et al., 1995). Antilisterial activ-ity of bacteriocins nisin and ALTATM2341 were tested by Szabo and Cahill (1998)under varying conditions of temperature and modified atmosphere. At 4°C and in aCO2-rich atmosphere, growth of L. monocytogenes was controlled in a broth medium.At 12°C (abuse temperature), addition of bacteriocins was necessary to preventgrowth of the pathogen. The authors expressed concern over the development of

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bacteriocin-resistant isolates and recommend investigation of resistance mechanismsprior to development of food applications for bacteriocins. Other considerationsinclude the following:

1. The factors affecting bacteriocin activity in foods. 2. Are bacteriocins active in the fresh-cut environment?

Natural Plant Volatiles

Volatile compounds not only comprise the aroma and flavor compounds of fruits andvegetables but may also play a functional role in plant-microorganism interactions.Interest in the use of these compounds is encouraged by consumer demands for“natural foods.” The natural origin of plant volatiles and the fact that they are con-sumed normally in fresh fruits and vegetables in the diet may enhance consumeracceptance of their use to control microbial spoilage. To date, interest in applicationof volatiles to fresh produce has focused on treatment of fruits to inhibit the growthof postharvest decay fungi. For example, Vaughn et al. (1993) analyzed volatiles fromraspberries and strawberry fruits, including benzaldehyde, 1-hexanol and 2-nonanone,for their ability to inhibit Alternaria alternata, Botrytis cinerea and Colletotrichumgloesporoides in vitro. Benzaldehyde inhibited all three fungi when added to growthmedia at 4 µl/mL. Similarly, Archbold et al. (1997) identified volatile compounds toinhibit the growth of Botrytis. Ten compounds including hexanal, 1-hexanol, methylsalicylate and methyl benzoate, prevented the growth of Botrytis on blackberries,strawberries and grapes. Volatiles from stone fruit origin including benzaldehyde(5000–10,000 ppm) and hexanal (2500 ppm) were shown by Caccioni et al. (1995)to exhibit a fungistatic effect against Monilia laxa and Rhizopus stolonifer.

Application of natural plant volatiles to fresh-cut salads to improve quality andsafety has been investigated by Dawson et al. (1999). In this study, a number ofvolatile compounds (acetic acid, ethanol and several pyruvates) were added to fresh-cut, mixed lettuce and cabbage to control the growth of pathogens such as L. mono-cytogenes and B. cereus. Acetic acid and the pyruvates reduced the total count ofthese vegetables by up to 2 log cycles, and populations of the pathogens were alsoreduced. Disadvantages of fumigation with volatiles include tainting effects resultingfrom compounds such as acetic acid, corrosive effects and toxic effects.

Nonvolatile compounds such as essential oils may also be used to inhibit spoilageflora and foodborne pathogens. Wan et al. (1998) found that washing lettuce with acomponent of basil oil (0.1–1.0% v/v) was as effective as washing with 125 ppm chlorine.The oil components were effective against Pseudomonas spp. and A. hydrophila.

INDUCED RESISTANCE

Plant tissues possess inherent defense mechanisms to enable resistance to microbi-ological invasion. Plant defense mechanisms and methods to induce resistance wererecently reviewed by Forbes-Smith (1999). To date, there are no published reportsexamining induced resistance and fresh-cut fruits and vegetables. The following sectionaims to introduce the concepts of plant defense mechanisms and induced resistance.

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Microbiology of Fresh-cut Produce 235

Synthesis of Phenolic Secondary Metabolites—Lignin and Phytoalexins

Lignification of tissues occurs to assist plants in resisting disease. Lignin is apolymeric polyphenol that combines with cellulose and pectin in the plant cell wallto increase resistance to pathogen penetration (Forbes-Smith, 1999). Phytoalexinsor phenolic “antibiotics” are also produced as a response to infection by microbialplant pathogens. An example is the phytoalexin glycinol, produced by soybeans.Glycinol was found to inhibit DNA, RNA and protein synthesis of the soft rotbacteria Erwinia caratovora (Weinstein and Albersheim, 1983).

Synthesis of Pathogenesis-Related Proteins

Some plant tissues produce antifungal proteins in response to invasion by microor-ganisms or as a response to exposure to ethylene (Schlumbaum et al., 1986; Enyediet al., 1992). For example, plant cells produce the lytic enzymes chitinase andchitosinase to degrade fungal cell walls (Baldwin et al., 1995).

Induced Resistance

It is possible to treat plant tissues with substances to elicit the natural defense mecha-nisms. Elicitors may be biotic (signal molecules produced by the plant) or abiotic(treatment with radiation such as UV radiation). Compounds such as methyl salicylateand chitosan are known to be antifungal and have been used to elicit resistance in fruits,including strawberries (Forbes-Smith, 1999). UV-C radiation has been used to treatprocessed carrots. Treatment with low-dose UV-C was found by Mercier et al. (1993aand b) to enhance resistance of the carrots to infection by Botrytis cinerea and Sclerotiniasclerotiorum at refrigeration temperatures. In a recent study, Lamikanra et al. (2002)reported the production of cyclic and acyclic terpenoid phytoalexins (β-ionone, gera-nylacetone, and terpinyl acetate) in cut cantaloupe melons exposed to UV radiation.Geranylacetone and terpinyl acetate, when added to cut cantaloupe (0.01% w/w),reduced microbial population from 6.2 × 108 in the untreated control to 1.2 × 108 and3.1 × 107 cfu/g, respectively, over a period of 24 h at 20°C. β-ionine completely inhibitedmicrobial growth under similar conditions. Application of induced resistance methodsto fresh-cut produce should be considered as an alternative biocontrol hurdle, as thecutting processes involved in fresh-cut processing may induce an elicitor response.Areas for future research include the effectiveness of elicitors in this environment andthe effects of treatment on the sensory quality of fresh-cut products.

CONCLUSION

Although the role of microorganisms in determining the safety and spoilage of fresh-cut produce is acknowledged in the literature, our understanding of the microbialecology is still limited. Optimization of processes to ensure freshness and safetyand the application of innovative biocontrol techniques will rely on fundamentalinvestigation of the growth, interactions and biochemical activity of associatedmicrobial species and the mechanisms underlying their development.

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Microbial Enzymes Associated with Fresh-cut Produce

Jianchi Chen

CONTENTS

IntroductionThe Microbial Enzymes

PectinasesCellulasesCutinasesProteinasesStarch-Hydrolyzing EnzymesLipolytic EnzymesOther Enzymes

The MicroorganismsThe Bacteria

The ErwiniaeThe PseudomonadsBacteria of Food Safety Concern The Lactic Acid Bacteria

The FungiThe Filamentous Fungi

BotrytisAspergillus

YeastsFurther Consideration References

INTRODUCTION

Plants harbor a diverse microflora ranging from the three primary domains of life:Bacteria, Archaea, and Eucarya (Andrews and Harris, 2000). Microorganisms onplant surfaces are also commonly associated with, or are contaminants from, soils,insects, mammals, and other animals (Jay, 1997). For agricultural produce, microbial

8

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contamination occurs at every stage of the production chain, from cultivation toprocessing. With the exception of plant pathogens, most microorganisms are gener-ally prevented from penetrating produce tissue by outer protective layers such asthe epidermis. Plant pathogenic microorganisms can directly attack plant tissues anddestroy the whole plant or plant parts. Fresh-cut produce presents an additionaldimension of the issue. Cutting destroys the internal cell compartment and createswounds on the plant organs. The wounded tissue releases plant juice or cell contentsthat serve as nutrients for microorganisms. Thus, cut surfaces, in most cases, areideal for the growth of microorganisms, including human pathogens.

Once in contact with plant, microorganisms are subjected to various levels ofmicrobial-plant interactions or interrelationships for their population expansion. Atthe low end, plants only provide a physical space or nutrients for microorganisms.At the higher level, plants and microbes recognize specific molecular signals fromeach other and trigger a series of biochemical reactions. The interaction depends onthe nature of the microorganisms, the host plant condition and the occurrence offavorable environmental factors. A response to these parameters could involve thecoordination expression of the microbial genes. With facultative plant pathogens,the interaction switches between the saprophytic and the pathogenic phases. Toparticipate in the microbe-plant interaction and explore the food sources, microor-ganisms may secrete enzymes, toxins, growth regulators and polysaccharides(Agrios, 1997), and the host plants may react to these substances accordingly. Amongthese substances, microbial enzymes have received the most attention.

Cell cytoplasm contains nutrients that are readily used by microorganisms (suchas simple sugars and amino acids) and carbon and energy reserves (such as starches,proteins and fats). A larger food source for microorganisms is plant biopolymers.Plant tissues are primarily cells and their metabolites. The undisrupted surface of afruit or vegetable is covered with a protective layer called cuticle consisting of cutins.Plant cell walls consist of cellulose, pectins and structural proteins. The middlelamella that is primarily pectins holds plant cells together. Inside a cell wall, cellmembrane consists of lipids and proteins. Microorganisms can convert cell polymersto soluble products that are transportable into the microbial cell. This degradationprocess is brought about by the action of one or more sets of enzymes secreted bymicroorganisms.

This chapter is not intended to be a comprehensive review of all microbialenzymes related to fresh-cut produce. In fact, few publications emphasize the per-spective of microbial enzymes associated with fresh-cut produce, although there area number of reviews related to the characteristics of microorganisms associated withproduce (Beuchat, 1996; Brackett, 1999; Nguyen-The and Carlin, 1994). Most com-monly, microbial enzymes were studied because of their roles in rot or spoilage ofvegetables and fruits. A more recent trend is the focus on a group of microorganismsthat are significant in terms of food safety (Liao et al., 1999). The chapter brieflydiscusses a few enzymes commonly produced by microorganisms related to fresh-cutproduce or wounded plant tissues. Because of the large volume of data, enzymesfrom plant pathogens have inevitably received more emphases despite of the fact thatmost of the plant microorganisms are not plant pathogenic. A wide range of enzymesare known to be involved in the degradation of plant cell polymers. These often include

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enzymes such as pectinases, cellulases, amylases, lypases and proteinases. Becauseof the interest in plant-pathogen interactions, and the industrial applications, pecti-nases have been extensively studied (Bateman and Miller, 1996; Barras et al., 1994;Blanco et al., 1999; Collmer and Keen, 1986; Herron et al., 2000; Hugouvieux-Cotte-Pattat et al., 1996; Lang and Dornenburg, 2000; Sakai et al., 1993).

THE MICROBIAL ENZYMES

P

ECTINASES

Pectic substances are one of the most abundant polysaccharides in cell walls ofhigher plants. They are composed primarily of linear polymeric chains of

D

-galacturonicacid linked as an

α

-1,4 glycoside that contains carboxyl groups either esterified(pectin) or nonesterified (pectic acid) to different degrees with methanol. Naturalpectins are present at various levels of methylesterification. Pectins are the basicconstituents of the intercellular cement in the middle lamella. Pectin degradationresults in liquefaction of the pectic substances leading to tissue maceration or softening.Pectic enzymes or pectinases degrade pectic substances. Degradation of pectic sub-stances is the main cause of rots in fruits or vegetables. Many microorganisms canutilize the monomers and oligomers of pectin as a food source. In the case of plantpathogens, the additional role of pectin degradation is its participation in the processof pathogenicity. Pectic enzyme activity enhances the capability of a pathogen topenetrate and colonize its hosts.

The complexity of the pectin biopolymer is indicated by the wide variety ofpectin-degrading enzymes available. Pectinases can be classified according to theirpreferential substrate: pectin or polygalacturonate, their reaction mechanism through

β

-elimination or hydrolysis, and the cleavage position in the polymer chain (

endo

-or

exo

-) (Sakai et al., 1993). In general, they are divided into two groups: pectinest-erases and depolymerizing enzymes. The pectinesterases or pectin methylhydrolase(EC 3.1.1.11) remove short branches of the pectin chains by deesterifying the methoxylgroup of pectin resulting in pectic acid. These enzymes have no effect on the overallchain length, but they may alter the solubility of the pectins and affect the activitiesof the enzymes in the second group. The second group of enzymes is the chain-splitting pectinases that cleave the pectinic chain and release shorter chain portionscontaining one or a few molecules of galacturonan. There are two subgroups. Onesubgroup involves enzymes that hydrolyze glycosidic linkages including polymeth-ylgalacturonase (PMG) and polygalacturonase (PG). Enzymes in the other subgroupcleave

β

-1,4 glycosidic linkages by transelimination, which results in galacturonidewith an unsaturated bond between C4 and C5 at the nonreducing end of the galactu-ronic acid formed. Similarly, two types of enzymes are in this subgroup: polymeth-ylgalacturonate lyase (PMGL) or pectin lyase and polygalacturonate lyase (PGL) orpectate lyase (PL). The properties and assays of pectic enzymes have been compre-hensively reviewed (Bateman and Miller, 1996; Barras et al., 1994; Sakai et al., 1993).

Production of pectolytic enzymes by microorganisms is known to be both consti-tutive and inductive. For plant pathogenic bacteria, the extracellular pectinases areregulated by the availability of the pectin polymer and the release of galacturonan units.

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A general model for pectinase induction is that the microorganism always producesa base-level amount of pectolytic enzymes. In the presence of pectin, this will releasea small number of galacturonan monomers, dimers or oligomers. These moleculesserve as inducers for enhanced synthesis and release of pectolytic enzymes. The increasein pectolytic enzyme concentration further increases the degradation of the pectinpolymer. After some time, however, high concentrations of generated monomers,dimers or oligomers then decrease the production of pectolytic enzymes. The pro-duction of pectolytic enzymes is also repressed when pathogens grow in the presenceof glucose (Agrios, 1997).

C

ELLULASES

Cellulose is a linear chain of glucose linked by

β

-1,4-glucosidic bonds up to severalthousand monomers. Because chains of six or more monomers are already insoluble,cellulose is insoluble in water. In general, enzymatic degradation of cellulose is aslow process. The major limiting factor in the hydrolysis of such materials isprobably the sequestration of single molecules of substrate by the enzymes involved(Warren, 1996). Fruits and vegetables contain much less cellulose relative to woodyplants. Cellulolytic enzymes secreted by pathogens play a role in the softening anddisintegration of cell wall material (Agrios, 1997). Cellulolytic enzymes may furtherparticipate indirectly in spoilage by releasing soluble sugars from cellulose chains.These soluble sugars can serve as food for the pathogen. Nonpathogenic microor-ganisms may also participate in cellulolysis for food resources.

Cellulases are commonly secreted by microorganisms to attack the cellulosepolymer. In addition to the common hydrolytic enzymes, oxidative and phosphoro-lytic enzymes are also involved in cellulose depolymerization (Warren, 1996). Thethree major types of hydrolytic cellulases that participate in the degradation ofcellulose to glucose are endoglucanases (

endo

-1,4-

β

-

D

-glucanohydrolase, EC 3.2.1.4)that randomly attack the cellulose chain and split the

β

-1,4-glucosidic bond; exo-glucanases (

exo

-1,4-

β

-

D

-glucan 4-cellobiohydrolase, EC 3.2.1.91) that release eithercellobiose or glucose from the nonreducing end of cellulose; and

β

-glucosidase orcellobiase (EC 3.2.1.21) that hydrolyze cellobiose and other water-soluble cellodex-trins to glucose (Singh and Hayashi, 1995). While fungi produce all of the threetypes of cellulases, all cellulolytic bacteria secrete a variety of endoglucanases (Beguin,1990).

C

UTINASES

The natural plant surface of leaves, flowers, fruits and young stems are covered bycuticle, which is a barrier protecting plants from pathogen invasion. The structuralcomponent of plant cuticle, called cutin, is an insoluble aliphatic biopolymer com-posed of hydroxy and hydroxyepoxy fatty acids. Many fungi and a few bacteria areable to produce cutinases. Fungal cutinase is composed of a single peptide with amolecular weight near 25,000 (Kolattukudy, 1985). As part of the pathogenicity,many fungal pathogens can penetrate the intact barriers (Kolattududy, 1985). With the

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production of cutinase, some fungi could grow on cutin as the sole source of carbon(Purdy and Kolattukudy, 1973).

P

ROTEINASES

In addition to its enzymatic function, proteins are constituents of cell membranesand structural components of plant cell walls. Proteinases or proteases catalyze thehydrolysis of peptide bonds in proteins or peptides. Proteinases produced by bacteriaand fungi are predominantly extracellular and can be classified into four groupsbased on the essential catalytic residue at their active site. They include serine proteases(EC 3.4.21), cysteine proteases (also called thiol proteases) (EC 3.4.22), aspartateproteases (EC 3.4.23) and the metalloproteases (EC 3.4.24) (Hase and Finkelstein,1994). Degradation of host proteins by proteinase secreted by microorganisms canprofoundly affect the organization and function of the host cells. However, fewinvestigations have been done on the nature and extent of degradation of plant tissues(Agrios, 1997). It has been documented that

Erwinia

spp. produces and secretesseveral proteases that have been associated with virulence in plants (Wandersmanet al., 1987).

S

TARCH

-H

YDROLYZING

E

NZYMES

Starch contains two kinds of glucose polymer:

α

-amylose and amylopectin. Theformer consists of long, unbranched chains of

D

-glucose in a unit connected by

α

-1,4glycosidic bonds. The glycosidic linkage of an amylopectin chain is

α

-1,4, but thebranch points are

α

-1,6 glycosidic bonds. Starch can be hydrolyzed to smaller unitsthat serve as nutrients for microorganisms. Starch hydrolysis requires enzymeshydrolyzing

α

-1,4 and, to a lesser extent,

α

-1,6 glucosidic bonds. The degradationof starch is brought about by the combined action of several types of enzymes calledamylases.

α

-Amylases cleave long starch molecules to oligosaccharides that are hydro-lyzed by glucoamylases,

β

-amylases, and other

exo

-

α

-1,4-glucanases (Nigam andSingh, 1995). The end product of starch breakdown is glucose that microorganismsuse directly.

L

IPOLYTIC

E

NZYMES

The common characteristic of all lipids is that they contain saturated or unsaturatedfatty acids. Oils and fats are found in many cells as energy storage compounds. Waxlipids are common on aerial epidermal cells. Phospholipids and glycolipids, alongwith proteins, are the main constituents of all plant cell membranes. Lipolytic enzymesor lipases hydrolyze lipids and liberate fatty acids. The microorganisms presumablyutilize the fatty acids directly (Agrios, 1997).

O

THER

E

NZYMES

The biochemical diversity suggests the potential of a large number of enzymes pro-duced by microorganisms. In addition to the enzymes mentioned above, some of theenzymes of importance in food production include anthocyanase, catalase, dextranase,

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glucose oxidase, invertase, lactase, etc. (Taylor and Richardson, 1979). In most cases,the roles of these enzymes in the microbe-host interaction remain to be studied.

THE MICROORGANISMS

Many microorganisms isolated from produce are known to produce extracellularenzymes. Magnuson et al. (1990) reported that approximately 10–20% of isolatesamong the mesophilic bacteria from shredded lettuce were pectinolytic. In manysamples of shredded carrots and shredded chicory salads, 20–60% of the isolatedpseudomonads were pectinolytic (Nguyen-The and Prunier, 1989). Recent investi-gations have revealed that sophisticated mechanisms often exist with plant pathogensthat can actively attack plant tissues for their population prosperity. One of the best-studied models is probably

Erwinia

species that cause soft rots of vegetables.In general, microorganisms on fresh-cut products fall in the category of bacteria,

yeasts and molds or filamentous fungi (Barriga et al., 1991; Lamikanra et al., 2000;Magnuson et al., 1990; Zagory, 1999). Yeasts and lactic acid bacteria are commonmicroflora on fruits. They do not actively attack plant tissues. However, they areresponsible for the spoilage of many fruit products, particularly in wounded or cuttissues where cell contents are released. Yeasts and lactic acid bacteria often usesimple sugars found in fruits to bring about fermentation, resulting in the productionof alcohol, organic acid and carbon dioxide. On the cut surface of fresh-cut produce,yeasts and lactic acid bacteria grow faster and often precede molds in the spoilageprocess. This was clearly demonstrated during an experiment of monitoring the micro-bial change on fresh-cut cantaloupe (Lamikanra et al., 2000). As spoilage progresses,degradation of the high molecular weight plant polymers is later brought about bymolds and other pectinolytic and cellulolytic bacteria (Jay, 1997).

T

HE

B

ACTERIA

Fresh vegetables are all subject to bacterial soft rots. The rotting process could beas short as three to five days under favorable conditions. Repulsive odor can usuallybe found with rotten cruciferous plants and onions. Through wounds, the soft rotbacteria enter plants and multiply quickly in the intercellular spaces. They producea set of enzymes such as pectinases, cellulases and proteases, dissolve the middlelamella and separate the cells. This causes maceration and softening of the affectedtissues. Several species in the bacterial genera of

Erwinia, Pseudomonas, Bacillus

and

Clostridium

are primary rotting pathogens of vegetables (Agrios, 1997). Therole of

Xanthomonas

in the soft rots of fruits and vegetables was also discussed byLiao and Wells (1987a). The soft rot bacteria can grow and are active over a rangeof temperatures from 5–35

°

C. They are killed with extended exposure at about 50

°

C(Agrios, 1997).

The Erwiniae

The soft rot Erwiniae produces large quantities of extracellular plant cell wall-degradingenzymes. Pectin methylesterases, pectate lyases, pectin lyase, polygalacturonase,cellulases, proteases and a phospholipase have been identified in

E

.

chrysanthemi

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(Collmer and Keen, 1986). Among all of these degrading enzymes, pectate lyaseshave a predominant role in plant-tissue maceration. Three soft rot erwinias,

E

.

carotovora

var.

carotovora,

E. carotovora

var.

atroseptica

and

E. chrysanthemi

havebeen extensively studied (Barras et al., 1994). The main characteristic distinguishingsoft rot erwinias from other

Erwinia

species is the ability to produce large quan-tities of pectic lyases. The enzyme macerates parenchymatous tissue of a widerange of plant species. The three soft rot erwinias have a worldwide distribution. Thehost range of

E. carotovora

var.

atroseptica

is mostly potatoes, a cool climate crop.

E. chrysanthemi

causes diseases in a wide range of tropical and subtropical crops.

E. carotovora

var.

carotovora

, however, has a wide distribution in both the temperateand tropical zones (Barras et al., 1994).

Wounds on the plant host are required for penetration of the soft rot bacteria.The pathogens feed and multiply on the plant juice from the wound surface. Theproduction of large amounts of pectolytic enzymes leads to further maceration ofthe tissues. The bacteria continue to multiply and advance in the intercellular spaces,while the surrounding plant cells plasmolyze, collapse and die. The invaded tissuessoon become soft with the appearance of a slimy mass consisting of innumerablebacteria swimming about in the liquefied substances (Agrios, 1997).

At the molecular level, the genes encoding pectic enzymes from penetratingpathogens are expressed in a characteristic manner in the infected tissue. The enzymesare exported from the pathogen cytoplasm to the host tissue milieu. Under favorableconditions, the enzymes are active and cleave structural polymers in the primarycell wall and middle lamella. The pectic fragments released by the enzymes mayalso have effects on the interaction, including the elicitation of host defense reactions.The results of the enzyme activity facilitate pathogen penetration, colonization andthe appearance of rot symptoms (Collmer and Keen, 1986).

Like

Escherichia coli,

Erwinia

species also belong the family of Enterobacteriacae.The genetic tools developed for the

E. coli

system could be applied to study

Erwinia

with minimum modifications. Thus,

E. chrysanthemi

and

E. carotovora

were selectedas model systems for the analysis of micro-plant interactions. These models havegenerated a great amount of data regarding the enzymes associated with soft rot diseases(Barras et al., 1994; Herron et al., 2000; Hugouvieux-Cotte-Pattat et al., 1996).

As summarized by Hugouvieux-Cotte-Pattat et al. (1996), the

E. chrysanthemi

strain 3937 produces five types of pectinases and multiple isoenzymes: at least sixendopectate lyases (PelA to PelE and PelL), an exopectate lyase (PelX), two pectinmethylesterases (PemA and PemB), a pectin lyase (PnlA) and an exopolygalactur-onase (PehX). While PelX, PemB and Ogl are intracellular enzymes, the otherpectinases are secreted into the extracellular medium by

E. chrysanthemi

cells.Among the six genes of endopectate lyases, five (

pelA, pelB, pelC, pelD

and

pelE

)play a major role in pectate lyase activity (Kotoujansky, 1987). These genes are orga-nized in two clusters:

pelB

and

pelC

in one cluster and

pelA, pelE

and

pelD

in another.The two clusters are widely separated on the bacterial chromosome (Hugouvieux-Cotte-Pattat et al., 1996). The PelL protein shows no homology with other pectinasesexcept for PelX, restricted to the C-terminal region (Brooks et al., 1990). In contrastto these endopectate lyases, which degrade long polymeric chains, oligogalacturonatelyase (Ogl) recognizes pectic oligomers of two to four residues. This cytoplasmic

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256

Fresh-cut Fruits and Vegetables: Science, Technology, and Market

enzyme cleaves the

α

-1,4 glycosidic bond by transelimination. The exopectate lyasePelX can utilize PGA and also methylated pectins as substrates (Brooks et al., 1990).It is expected that this type of enzyme is present in the bacterial periplasm because itacts better on oligomers produced by endopectate lyases than on polymeric substrates.Most soft rot Erwiniae produce an endopectin lyase (PnlA) activity in response toDNA-damaging agents (Tsuyumu and Chatterjee, 1984).

In constrast to lyase, hydrolase does not appear to be the predominant pectindepolymerase in

E. chrysanthemi

. An exo-cleaving polygalacturonase (PehX), theonly hydrolase, was found in

E. chrysanthemi

. The gene

pemA

, which encodes anextracellular pectin methylesterase, is linked to the

pelA, pelE, pelD

locus encodingthree major pectate lyases (Kotoujansky, 1987). The gene

pemB

, which encodes anovel pectin methylesterase, is an outer membrane lipoprotein. The activity of thisenzyme is approximately 100-fold higher on pectic oligomers than on natural pectins.The action of extracellular pectinases on pectin probably liberates small methylatedoligogalacturonides that can enter the periplasm by diffusion, and the role of

pemB

might be to degrade such oligomers (Hugouvieux-Cotte-Pattat et al., 1996).Cellulases produced by

E. chrysanthemi

were secreted to the culture medium.Mutation in the secretory machinery resulted in the accumulation of cellulase andpectinase in the periplasmic space (Andro et al., 1984). The genetic locus of the secretorymachinery was later determined to consist of 15 genes organized in five transcrip-tional units (Salmond, 1994) to form a type II secretion machinery. The genes oftwo cellulases, CelY (EGY) and CelZ (EGZ) were later cloned and sequenced(Guiseppi et al., 1988, 1991). The two genes do not appear to be homologous. WhileCelZ represents aproximately 95% of the total carboxymethyl cellulase activity,synergistic hydrolysis was observed from the two enzymes (Zhou and Ingram, 2000).Synergy did not require the simultaneous presence of both enzymes. But, it isimportant that CelY was used as the first enzyme (Zhou and Ingram, 2000).

The export pathway of proteinases is different from those of cellulases andpectinases. The extracellular state of an

E. chrysanthemi

protease was not affectedby the Out mutation(s) (Andro et al., 1984). Wandersman et al. (1987) studied threeclosely related metalloproteases—A, B and C. They localized the structural genesfor proteases B and C (prtB and prtC) as two distinct adjacent transcription units.Three genes,

prt

D,

prt

E and

prt

F, are needed for the specific secretion of the prtB-and prtC-encoded proteases. The prtD, prtE and prtF genes are part of an operonlocated immediately upstream of

prt

B and

prt

C. Expression of these genes in

Escherichia coli

allows the specific and efficient secretion of protease B and proteaseC (Letoffe et al., 1990). Marits et al. (1999) isolated an extracellular protease gene(

prt

W) from a strain of

E. carotovora

subsp.

carotovora

. The prtW gene was foundto be strongly induced in the presence of plant extracts. An insertion mutation inthis gene exhibited reduced virulence, indicating that the proteinase gene plays animportant role in maceration of plant tissue.

The Pseudomonads

The production of pectolytic enzymes by pseudomonads that are involved in mac-eration of host tissues is mainly the nonfluorescent species such as

P. cepacia, P.caryophylli

and

P. gladioli

(Gross and Cody, 1985). These enzymes are secreted

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Microbial Enzymes Associated with Fresh-cut Produce

257

directly into the plant tissue and cause rotting. Endo-polygalacturonase was reportedto be the principal enzyme produced during infection of P. cepacia (Ulrich, 1975).Tissue degradation resulted in the decrease of pH from 5.5 to as low as 4.0. Sucha lower pH value is favorable to the high endo-polygalacturonase activity. It is alsowell known that a fluorescent species, P. marginalis causes soft rots of variousvegetables. However, pectate lyase (polygalacturonate trans-eliminase is responsiblefor maceration of host tissues (Zucker et al., 1972). In contrast to that of P. cepacia,the infection from P. marginalis led to the increase of pH value, corresponding tothe high pH optima of the pectate lyase. In addition, P. cepacia also produces apectate lysase (Ulrich, 1975). Pseudomonas cepacia is currently under the name ofBurkholderia cepacia.

Among the 128 strains of pectolytic bacteria studied, Liao and Wells (1987b)identified 55 strains (43%) as fluorescent Pseudomonas spp., only second to Erwiniacarotovora (64 strains or 50%). More importantly, the Pseudomonas strains couldgrow at 4°C. The psychrotrophic characteristics of the pectolytic Pseudomonasundoubedly represent a threat to fresh-cut produce, because the usual recommendationis for fresh-cut produce to be stored under low temperature conditions. Psychrotrophicpseudomonads have been known to cause spoilage in vegetables stored at lowtemperatures (Brocklehurst and Lund, 1981; Magnusson et al., 1990; Nguyen-The andPrunier, 1989). Only a few pseudomonads have been identified to produce cellulasescompared to pectolytic enzymes (Gross and Cody, 1985). Cellulase activity was notdetected in all of the Pseudomonas strains investigated by Liao and Wells (1987b).

Bacteria of Food Safety Concern

The pectic enzymes from foodborne human pathogenic bacteria are becoming thefocus of some researchers. The pectic enzymes from these bacteria are not extracel-lular (Chatterjee et al., 1979). Knowledge about pectic enzymes from thesesaprophytic but human pathogenic bacteria becomes more relevant with the growthof the fresh-cut produce industry. These bacteria can utilize the readily availablepectic substrates of plant produce for growth and survival purposes.

Pectic enzymes from Yersinia species have received some investigation. The firstreport came from Von Riesen (1975), who observed that the human and animalpathogenic Y. enterocolitica and Y. pseudotuberculosis could digest polypectate gel.Later, Starr et al. (1977) demonstrated the production of pectate lyase from Yersiniaspecies. The intracellular pectate lyase was then further characterized (Bagley andStarr, 1979). In strains of Y. enterocolitica and Y. pseudotuberculosis, pectate lyaseis a periplasmic and cytoplasmic enzyme. In Klebsiella pneumoniae, another speciesof enteric bacteria, pectate lyase is entirely cytoplasmic. These contrast to the softrot Erwinia species, where a large quantity of extracellular pectate lyase is producedand almost totally excreted. A much higher level of pectate lyase activity wasdetected in cells of K. pneumoniae, but not culture supernatants, when grown onpolygalacturonate than when grown on other carbon sources, indicating the catabolicfunction of pectic enzymes in this bacterium (Chatterjee et al., 1979).

Liao et al. (1999) recently characterized an exopolygalacturonase and a pectatelyase from Y. enterolitica. A 1803 bp gene of a polygalacturonase pehY was cloned.

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258 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Purified polygalacturonase was unable to macerate plant tissue. The deduced aminoacid sequence of pehY showed 59% identity to the exopolygalacturonase (exoPG)of E. chrysanthemi (He and Collmer, 1990) and 43% identity to the exopolygalac-turonase of Ralstonia solanacearum (Huang and Allen, 1997). The polygalactur-onase was determined to be exolytic, whereas the pectate lyase was endolytic. BothpehY and pel genes of Y. enterolitica are possibly encoded in the chromosome ratherthan plasmid-borne.

The Lactic Acid Bacteria

The lactic acid group of bacteria is loosely defined with no precise boundaries. Acommon characteristic that they all share is the production of lactic acid from hexosefermentation. Bacteria in this group do not have functional Krebs cycle and heme-linked electron transport systems or cytochromes. They obtain their energy bysubstrate-level phosphorylation (Jay, 1997). The common genera of lactic acid bac-teria include Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconos-toc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus andWeissella (Stiles and Hozapfel, 1997). Lactic acid bacteria are widely used in foodbiotechnology, including food or feed production from raw plant material. Karamand Balarbi (1995) reported that polygalacturonases and pectin esterases were alsopresent in lactic acid bacteria. Lactic acid bacteria are not necessarily destructive toplant tissue. The ability of lactic acid bacteria to alter food flavor is, however, wellknown. A possible pathway of fruit flavor deterioration by lactic acid bacteria is byway of an increased lipase production (Chandler and Ranganathan, 1975; Meyerset al., 1996). The delicate balance of flavors in fruits could be more severely affectedby the growth of lactic acid bacteria than vegetables, and this might contribute tothe relatively rapid flavor loss in minimally processed fruits (Lamikanra et al., 2000).

In a recent investigation, Lamikanra et al. (2000) reported the decay of fresh-cut cantaloupe stored at 20°C with the dominant microflora being Gram-positivebacteria, and the concurrent increase in lactic acid production. However, it is gen-erally believed that lactic acid bacteria do not directly attack plant cell wall polymers.At 4°C, fruit stored did not show significant degradation over 14 days. Gram-negativerods were the dominant microflora with no lactic acid present. The Gram-negativebacteria were believed to be psychrotrophic pseudomonads. These pseudomonadsare capable of producing pectic enzymes that would have been expected to degradethe fruit tissue. The observation is an indication of the need for further research onthe effects of microorganisms on fresh-cut fruit tissue surfaces.

THE FUNGI

Fungi can produce a wide array of enzymes, such as amylase, protease, lipase,cellulase and pectinase, that degrade plant polymers. From the industrial applicationpoint of view, Aspergillus oryzae and Aspergillus niger, together with a bacterium,Bacillus subtilis, are the three most useful, well-known and safe microbial sourcesfor enzymes (Talyor and Richardson, 1979). Pectic enzymes have received consid-erable attention regarding their involvement in fruit and vegetable rots.

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Microbial Enzymes Associated with Fresh-cut Produce 259

The role of pectic enzymes in plant-fungi interaction was actually postulatedmore than a century ago by De Bary in 1886 (Lang and Dornenburg, 2000). Manyfilamentous fungi such as Aspergillus, Cercospora, Fusarium, Penicillium, Rhizoc-tonia and Trichoderma are known to produce large amounts of extracellular pecticenzymes. Although yeasts are not considered to be capable of actively attackingplant tissue, the production of pectic enzymes by yeasts has long been known. In1951, Luh and Phaff first described pectic enzymes in Saccharomyces fragilis. Sincethen, a few yeast species have been reported to produce pectic enzymes (Blanco et al.,1999).

The Filamentous Fungi

The degradation process of plant polymers by filamentous fungi involves the actionof a number of extracellular pectic enzymes. In contrast to the widespread occurrenceof endopectate lyase in bacteria, fungi mostly produce polygalacturonases and pectinesterases (Sakai et al., 1993). Most fungal polygalacturonases are endoenzymes.However, some fungi also produce exoenzymes. Polygalacturonase genes have beendescribed in a large number of phytopathogenic and nonphytopathogenic fungi,including some of the fruit rot species in Aspergillus, Botrytis, Colletorichum andPenicillium. In most of these cases, a detailed genetic analysis reveals the existenceof polygalacturonase gene families rather than a single polygalacturonase gene (Langand Dornenburg, 2000). The synthesized pectinases are generally secreted fromintact cells into the surrounding tissue. However, some enzymes might remain insidethe cell, obviously for their catabolic function. In a comparative study, the Penicilliumfrequentans synthesized 11 polygalacturonases and two pectinesterases when grownin liquid culture supplemented with pectin. Seven polygalacturonases and the twopectinesterases were secreted in the medium, whereas four polygalacturonases werenot secreted (Kawano et al., 1999).

In a study with grape bitter rot caused by Greenaria uvicola, Ridings and Clayton(1970) related the production of pectic enzymes by the pathogen to the rot symptom.G. uvicola produced polygalacturonase in four- and seven-day-old cultures andpectate lyase (trans-eliminase) in 12-day-old cultures. Pectin methylesterase or cel-lulolytic enzymes were absent. They further found that G. uvicola is not restrictedin its pathogenicity to the fruits of Vitis spp. Based on the inoculation experimentthrough wounded fruits or vegetables, they reported that G. uvicola induces rots ofseveral fruits, including peach, apple, strawberry, banana and blueberry. The impor-tance of pectolytic enzymes for fungal virulence was further demonstrated by themutagenesis experiments of endopolygalacturonase genes in Aspergillus flavus(Shieh et al., 1997) and Botrytis cinerea (Ten Have et al., 1998). The inactivationsof these genes were directly associated with the loss of virulence.

It should, however, be noted that not all of the PG genes are involved in virulence.The regulation of the polygalacturonase gene expressions is apparently of great impor-tance on the enzyme biological function. Similar to the bacterial counterpart, pecticenzymes in fungi are produced both inductively and constitutively. Whitehead et al.(1995) reported that the polygalacturonase genes of Aspergillus flavus were inducedin pectin-containing media but not in glucose. The regulation is at the transcriptional

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260 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

level, suggesting that endopolygalacturonases participate in host penetration bydegrading the pectin layer.

Botrytis Botrytis cinerea is an important pre- and postharvest pathogen of many fruits. Inthe early 1900s, several biochemical studies showed that endopolygalacturonaseproduction by B. cinerea was affected by the carbon source available, particularlyin the presence or absence of pectin in the culture medium (Johnston and Williamson,1992; Leone, 1990). The endopolygalacturonase genes from B. cinerea were latercloned (Ten Have et al., 1998; Wubben et al., 1999). B. cinerea produces a set ofendopolygalacturonase isozymes. The different endopolygalacturonase isoforms ofB. cinerea are encoded by a gene family of at least six genes (Bcpg1–6) (Ten Have et al.,1998; Wubben et al., 1999). The expression of the different endopolygalacturonase-encoding genes of B. cinerea was affected in liquid culture by the carbon sourceavailable (Wubben et al., 1999) as well as by changes in pH of the culture medium(Wubben et al., 2000). A basic constitutive expression level was observed for twogenes, Bcpg1 and Bcpg2 which encode basic isozymes. Galacturonic acid was shownto induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resultedin induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was induc-ible; however, the inducing factors could not be identified. There is evidence thatBcpg5 gene expression is favored by a combination of low pH and galacturonic acidinduction. Finally, galacturonic acid-induced expression of the Bcpg4 gene wasrepressed by the presence of more favorable carbon sources, such as glucose(Wubben et al., 2000).

Urbanek and Kaczmarek (1985) reported that an apple strain of B. cinereaproduced extracellular acid proteinases, aspartic proteinase and carboxypeptidase.They noted that isolated aspartic proteinase hydrolyzed proteins in the preparationsof apple cell walls and that the excretion of aspartic proteinase preceded that ofcarboxypeptidase. Doss (1999) analyzed the composition and enzymatic activity ofthe extracellular matrix secreted by germlings of B. cinerea to serve in part in theirattachment. Cellulase, pectin lyase and pectin methylesterase activities were noted,but proteinase activity was not detected.

AspergillusMost of the currently used pectinolytic enzymes in food processing are derived fromAspergillus species. A. niger is probably one of the best analyzed fungi with respectto polygalacturonase synthesis. It possesses a complete family of endopolygalactu-ronase encoding genes and produces several polygalacturonase isoenzymes thatdisplay considerable differences with respect to substrate specificity, cleavage rateand optimal pH for activity (Parenicova et al., 1998). Polygalacturonases are abun-dant among the saprophytic fungi using dead plant tissue as a food source. They aremostly present together with other pectinases and are the enzymatic machinery fordegrading the complex plant material and their conversion to readily metabolizablecarbohydrates. A. niger also produces other plant cell wall degradation enzymes.Gherbawy (1998) surveyed the presence of fungi on plums, pears and apples inEgypt. A. niger was found to be the dominant fungus. He also reported that the low

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Microbial Enzymes Associated with Fresh-cut Produce 261

dose of gamma irradiation (1 Mci for 10 min) enhanced the A. niger isolates toproduce more biomass and polygalacturonase, pectinmethylesterase, cellulase andprotease.

For plant pathogenic species, it is clear that pectic enzymes are involved inpathogenicity or virulence. A. flavus harbors two glucose-repressible and one con-stitutive endopolygalacturonase gene (Cleveland and Cotty, 1991). The gene pecAwas shown to be related to pathogenicity. In another study (Shieh et al., 1997), theinvolvement of one of the endopolygalacturonases (P2c) in fungal aggressivenesswas positively demonstrated by using genetic manipulation methods. Deleting theP2c gene significantly decreases the ability of the fungus to spread in cotton balls,while introducing the specific gene into a P2c null mutant increased the aggressive-ness of the strain. It is interesting to note that in the nonpathogenic A. nidulans, aphytopathogenic potential could be demonstrated under conditions when polygalac-turonase synthesis was induced (Dean and Timberlake, 1989).

YEASTS

Although yeasts are common microflora of plant surface, particularly on fruits, theyare not plant pathogens. Yeasts are highly efficient in metabolizing simple sugars.When the cell content is released, yeasts can multiply quickly in fruit juice by thefermentation of simple sugars. Therefore, yeasts play an important role in fruitspoilage under favorable conditions. Pectic enzymes have been reported in severalyeast species. These yeasts belong to the genera Canidia, Cryptococcus, Fabospora,Kluyveromyces, Pichia, Saccharomyces and Zygosaccharomyces (Blanco et al.,1999). With the exception of a few cases, pectic enzymes from yeasts are mainlyendopolygalacturonases (Gainvors et al., 1994). The end products of endopolyga-lacturonases reactions are always oligosaccharides with a varying number of galac-turonic residues. Moreover, all of these enzymes preferentially attack pectate overpectin, and their activities decrease as the degree of methylation increases (Blancoet al., 1999). Barnby et al. (1990) found that the activity of endo-polygalacturonasefrom K. marxianus with 37.8% esterified pectin is about 95%, and with 61% pectinesterification, the activity decreases to 25%.

The function of pectic enzymes in yeasts is largely unknown. Blanco et al. (1999)proposed two possible functions. For the endopolygalacturonase producing group,most authors have attributed an ecological role rather than a trophic one to yeastpolygalacturonases. These enzymes could be involved in substrate colonization onfruits, causing the breakdown of plant tissues with a concomitant release of sugarsfrom plant cells. This, in turn, can be utilized for yeast growth and can, consequently,cause further spoilage. The other group encompasses the yeasts that, like filamentousfungi, have the ability to grow using pectic substances as the sole carbon source(Federici, 1985).

The production of pectic enzymes by yeasts is usually constitutive. However,the pectolytic capacity of a few species such as C. albidus has been reported to beinducible (Blanco et al., 1999). Cryptococcus albidus, together with some filamen-tous fungi, was reported to produce an inducible endopolygalacturonase that isinvolved in the spoilage of preserved fruit (Federici, 1985).

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262 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Using classical genetic techniques, Blanco et al. (1997) demonstrated that thepectolytic capacity of S. cerevisiae had at least two structural genes in the wild-typestrain 1389, whereas the genetic strain IM108b was monogenic. Iguchi et al. (1997)reported the cloning of a protopectinase-encoding gene (PSE3) from Trichosporonpenicillatum. The PSE3 gene contains an ORF encoding a 367 amino acid protein.The deduced amino acid sequence for this gene shows a high homology (65.4%)with the polygalacturonases from Aspergillus oryzae and other filamentous fungi.

FURTHER CONSIDERATION

It is evident that further research on the effects of microorganisms and microbialenzymes on plant tissue as they relate to fresh-cut produce is needed. The presenceof a large area of wound surface or large number of wounded cells and the refrig-eration requirements are factors for consideration. In fruits, for example, flavorchanges occur rapidly regardless of refrigeration. Although the cause of such changesin flavor is a subject for research, activities of pectic enzymes as well as otherdepolymerases could contribute significantly enough to cause changes in flavor.Microorganisms are usually considered to be the source of these enzymes. Theprecise biological role of some depolymerases such as polygalacturonases, however,remains unclear even in the tomato fruit, which has been extensively studied (Langand Dornenburg, 2000). It is also possible that deterioration caused by other non-microbial physiological changes precedes microbial activity. Such changes could beresponsible for high microbial activities that further affect quality. Thus, it is con-ceivable that physiological changes, including nonmicrobial changes that degradecells, could precede an increase in microbial activity (Zagory, 1999). Increasedmicrobial activity could then be the result of the contents of the ruptured cells servingas substrates for microorganisms. In such cases, the presence of the microbialenzymes is the result, and not the cause, of spoilage. However, enzymes producedthrough this pathway would be expected to further degrade produce quality. Regard-less of the mode of product deterioration, it is evident that interactions betweenmicroorganisms and the plant host play significant roles in the quality of fresh-cutfruit and vegetable products. Knowledge of the microbe-plant interactions in fresh-cutproduce will help to develop strategies to improve their sensory quality and shelf life.

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Gross, D. C. and Cody, Y. S. 1985. “Mechanisms of plant pathogenicity by Pseudomonasspecies.” Can. J. Microbiol. 31:403–410.

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Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W., and Reverchon, S. 1996. “Regula-tion of pectinolysis in Erwinia chrysanthemi.” Annu. Rev. Microbiol. 50:213–257.

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lacturonases from Botrytis cinerea.” Mycol. Res. 96:343–349.Karam, N. E. and Balarbi, A. 1995. “Detection of polygalacturonases and pectin esterases in

lactic acid bacteria.” World J. Microbiol. Biotechnol. 11:559–563.Kawano, C. Y., Chellegatti, M. A. S. C., Said, S., and Fonseca, M. J. V. 1999. “Comparative

study of intracellular and extracellular pectinases produced by Penicillium frequen-tans.” Biotechno. Appl. Biochem. 29:133–140.

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Preservative Treatments for Fresh-cut Fruits and Vegetables

Elisabeth Garcia and Diane M. Barrett

CONTENTS

IntroductionFresh-cut Products and Color Preservation

Enzymatic BrowningPreharvest Factors Postharvest and Processing FactorsBrowning and Enzymes Other Than Polyphenoloxidase

Control of Enzymatic BrowningAntibrowning Agents

AcidulantsReducing AgentsChelating AgentsComplexing AgentsEnzyme Inhibitors Other Antibrowning Agents Application of Antibrowning Agents Combined Treatments

Physical Treatments and Browning ControlReducing Oxygen AvailabilityReducing TemperatureApplying Gamma RadiationUse of Other Nonthermal Technologies

Other Color ChangesWhite Blush in Carrots Yellowing or Degreening

Prevention of Texture Loss in Fresh-cut ProductsFruit and Vegetable Tissue Firming

Calcium and/or Heat TreatmentsUse of Modified Atmosphere Packaging

Water Loss Prevention

9

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Appendix: Evaluation of Enzymatic BrowningReflectance Measurements Browning Index Loss of ReflectanceEstimate of Apple Susceptibility to BrowningVisual Evaluation of Browning

References

INTRODUCTION

From the quality standpoint, it is desirable to preserve the characteristics of fresh-cut fruits and vegetables at their peak. What the consumer perceives as the mostappealing attributes of these products include their fresh-like appearance, taste andflavor, in addition to convenience. Obviously, any food product should be safe forconsumption, and fresh-cut products are very sensitive to contamination. Among thelimitations to shelf life of fresh-cut products are microbial spoilage, desiccation,discoloration or browning, bleaching, textural changes and development of off-flavoror off-odor. Nevertheless, safety aspects are not discussed in this chapter, but werereviewed in Chapter 4. The primary quality attributes of a food product include color,texture, flavor and nutritional value. When assessing plant product quality, consumerstake product appearance into consideration as a primary criterion, and color is prob-ably the main factor considered (Kays, 1999).

While conventional food-processing methods extend the shelf life of fruits andvegetables, the minimal processing to which fresh-cut fruits and vegetables aresubmitted renders products highly perishable, requiring chilled storage to ensure areasonable shelf life. Preparation steps such as peeling or scrubbing, slicing, shred-ding, etc., remove the natural protection (peel or skin) of fruits and vegetables andcause bruises, rendering them susceptible to desiccation and wilting. This also exposesinternal tissues to microbes and potentially deleterious endogenous enzymes. Amongthe possible consequences of mechanical injuries to produce are increase in respi-ration rate and ethylene production, accelerated senescence and enzymatic browning(Rosen and Kader, 1989). In conventional types of fruit and vegetable processing,such as canning and freezing, many of these problems are prevented or controlledby heat processing and consequent inactivation of enzymes by the use of protectivepackaging materials, or through the application of various additives. In the productionof fresh-cut products, the use of heat is avoided in order to prevent cooking of theproduct and, consequently, loss of fresh-like characteristics. Several chemical pre-servatives can be used, depending on what is to be prevented; often, chemicalpreservatives are applied in the control of enzymatic browning, firmness and decay(Brecht, 1995). Other important applications include the use of controlled modifiedatmosphere packaging, and edible films also have many potential applications.

A survey on consumer perception of convenience products revealed the desirethat such products maintain fresh characteristics longer without the use of preser-vatives (Bruhn, 1994). Unfortunately, depending on the type of quality defect to beprevented or controlled, it is not always possible to avoid the use of chemical treat-ments. One important aspect to consider is the establishment of conditions that allow

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for quality optimization at a reasonable shelf life, rather than extending shelf life atan acceptable quality (Shewfelt, 1994).

In this chapter, we review the most common treatments used to preserve thecolor and texture of fresh-cut products. Color preservation is, after safety, the mostimportant attribute to be preserved, because frequently, a product is selected for itsappearance, particularly its color. Color has been considered to have a key role infood choice, food preference and acceptability, and may even influence taste thresh-olds, sweetness perception and pleasantness (Clydesdale, 1993). Second, texture lossand preservation in fresh-cut products will be discussed, due to its important impacton product appearance and sensory quality.

FRESH-CUT PRODUCTS AND COLOR PRESERVATION

Fruits and vegetables are attractive and eye-catching to a large degree because ofthe richness of pigments that they contain. Preservation of chlorophyll in vegetables,red to purple anthocyanins, and yellow, orange and red carotenoids in fruits andvegetables is of vital importance to maintain quality. Color changes (Figure 9.1) infresh-cut fruits and vegetables may have different origins, for example, decreasedgreen pigmentation in fresh-cut lettuce may result from senescence, heat exposureor acidification; discoloration or browning of sliced mushrooms, apples and pearsis brought about through the action of polyphenol oxidases; and white blush devel-opment in carrots is initially caused by desiccation and later, by lignification. Themain focus of this chapter is on prevention of enzyme-catalyzed browning, althoughsome of the other color changes will be briefly discussed.

E

NZYMATIC

B

ROWNING

Enzymatic browning is one of the most limiting factors on the shelf life of fresh-cut products. During the preparation stages, produce is submitted to operations wherecells are broken, causing enzymes to be liberated from tissues and put in contactwith their substrates. Enzymatic browning is the discoloration that results from theaction of a group of enzymes called polyphenol oxidases (PPOs), which have beenreported to occur in all plants and exist in particularly high amounts in mushroom,banana, apple, pear, potato, avocado and peach. Enzymatic browning must be dis-tinguished from nonenzymatic browning, which results upon heating or storage afterprocessing of foods. Types of nonenzymatic browning include the Maillard reaction,caramelization and ascorbic acid oxidation.

Enzymatic browning is a complex process that can be subdivided in two parts. Thefirst part is mediated by PPO (Figure 9.2), resulting in the formation of

o

-quinones(slightly colored), which through nonenzymatic reactions, lead to the formation of complexbrown pigments.

o

-Quinones are highly reactive and can rapidly undergo oxidation andpolymerization.

o

-Quinones react with other quinone molecules, with other phenolic com-pounds, with the amino groups of proteins, peptides and amino acids, with aromatic amines,thiol compounds, ascorbic acid, etc. (Whitaker and Lee, 1995; Nicolas et al., 1993).Usually, brown pigments are formed, but in addition, reddish-brown, blue-gray and evenblack discolorations can be produced on some bruised plant tissues. Color variation in

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products of enzymatic oxidation is related to the phenolic compounds involved in thereaction (Amiot et al., 1997), and both color intensity and hue of pigments formed varywidely (Nicolas et al., 1993). Consequences of enzymatic browning are not restric-ted todiscoloration—undesirable tastes can also be produced, and loss of nutrient quality mayresult (Vámos-Vigyázó, 1981). Biochemical details on PPO action were reviewed inChapter 6. PPO has been considered one of the most damaging enzymes to qualitymaintenance of fresh produce (Whitaker and Lee, 1995), and the prevention of enzymaticbrowning has always been considered a challenge to food scientists (Ponting, 1960).

Preharvest Factors

Several parameters may contribute to the development of enzymatic browning.Agricultural practices, soil, fertilizers, climate and harvesting conditions all affectthe final quality of fresh-cut products (Ahvenainen, 1996). High nitrogen levels havebeen related to a greater tendency to brown in potatoes (Mondy et al., 1979).

FIGURE 9.1

Some examples of changes in appearance of cut fruits and vegetables. Browningis shown in photos A–G. (A) Banana slices after 0,1,2, and 3 days of cutting. (B) Applewedges; left: immediately after cutting, right: 24 hr after cutting. (C) Comparison of browningdevelopment among three pear varieties: ‘Red D’Anjou’ (left), ‘Hi-Up’ (center), ‘Bartlett’(right). (D) Avocado halves. (E) Potato sticks. (F and G) Romaine lettuce. (H) White blush(right) in baby carrots. (I) Wrinkling on green bell peppers (right).

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

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The selection of raw material for processing needs to be carefully evaluated. Thesusceptibility to browning may differ from cultivar to cultivar, as exemplified in Tables 9.1and 9.2. Some tissues may have high PPO activity and/or high concentration or typesof phenolic PPO substrates which, under appropriate conditions, lead to a highertendency to brown. In pears, it was found that although the phenolic content tendedto decrease with delayed harvest time, phenolic levels did not always correlate withthe susceptibility to browning (Amiot et al., 1995). In general, high concentrations

TABLE 9.1 Enzymatic Browning in Purées Prepared with Various Apricot Cultivars at Commercial Maturity

Cultivar DL

*

‘Henderson’ 26.3‘Moniqui’ 21.4‘Rouge de Roussillon’ 17.8‘Rouge de Fournes’ 17.8‘Polonais’ 16.8‘Canino’ 16.7‘Cafona’ 11.8‘Bebeco’ 5.3‘Precoce de Tyrinthe’ 3.7

DL* = Difference in lightness between oxidizedand nonoxidized apricot purées.

Source:

Adapted from Radi et al., 1997.

FIGURE 9.2

Reactions that can be catalyzed by polyphenol oxidase: (1) hydroxylation ofmonophenols to

o

-diphenols and (2) oxidation of

o

-diphenols to

o

-quinones.

OH

+ 1/2 O2

R

PPO

OH

O

R

O

=

=+ H2O

+ AH2

OH

R

+ O2

PPO

R

OH

OH

+ A + H2O(1)

(2)

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of phenolic compounds are found in young fruits. While in bananas, PPO activityis higher in the pulp than in the peel, in pear and apple, PPO activity is higher in thepeel than in the flesh (Macheix et al., 1990). In addition, PPO activity may varywidely between cultivars of the same crop and at different maturity stages. Examplesof such variations are shown in Table 9.3. Ideally, produce varieties with either lowlevels of PPO or phenolic substrates, or both, should be selected for fresh-cut process-ing. New varieties with desirable traits for fresh-cut processing may be developed byconventional breeding techniques and potentially through biotechnology (Chapter 13).Nevertheless, it is important to point out that not only PPO activity and concentrationof substrates are important, but also, individual phenolics exhibit different degrees ofbrowning, and the rate of enzymatic browning is also affected by other polyphenolcompounds present in the tissue (Lee, 1992).

Postharvest and Processing Factors

Processing operations such as washing, scrubbing, peeling, trimming, cutting, shred-ding, etc., carried out during the initial stages of fresh-cut preparation cause mechanicalinjury to the plant tissues. Moreover, even prior to processing, produce manipulationmay bring mechanical shocks resulting in cracks and bruises, which can elicit phys-iological and biochemical responses in the wounded tissue as well as in unwoundeddistant cells (Saltveit, 1997). Peel removal and loss of tissue integrity with cell

TABLE 9.2Susceptibility of Potato Varieties to Enzymatic Browning after Storage (of Whole Unpeeled Tubers) at 5

°

C and 75% RH

Browning Index

Storage Time var. ‘Bintje’ var. ‘Van Gogh’ var. ‘Nicola’

1 mo.30 min* 6 27 4460 min 15 40 75120 min 21 52 88

5 mo.30 min 16 26 2160 min 23 56 58120 min 30 78 98

8 mo.30 min 10 28 6660 min 32 74 112120 min 62 104 145

Note:

Browning evaluation was carried out on 5 mm slices cut from thecenter of the tubers and left at 23

°

C for observation at 30 min, 60 minand 120 min after cutting.

Source:

Adapted from Mattila et al., 1993.

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breakage facilitate microbial contamination. In addition, exposure to air and releaseof endogenous enzymes that are put in contact with their substrates, originally indifferent cell compartments, may lead to detrimental consequences. Living tissuesare still physiologically active and respond to wounding. The first responses tomechanical injury relate to respiration rate increase and possibly increased ethyleneproduction (see Chapter 5). In general, respiration rates are inversely related to theshelf life of produce. Quality deterioration may result from increased ethylene pro-duction, which may induce higher cellular metabolism and higher enzymatic activity(Reyes, 1996). Another consequence of wounding is the induction of secondaryproduct synthesis, including a variety of phenolic compounds. Among the enzymesthat may have deleterious effects, polyphenol oxidase (PPO) can be the most dam-aging enzyme with regard to color deterioration of plant foods (Whitaker and Lee,1995).

During peeling and cutting operations, if the equipment used is not in the bestcondition, for example, if dull knives and blades are used, bruising and damageoccurs in more tissue layers than intended; thus, the sharpness of knife blades cansignificantly affect product storage life (Bolin et al., 1977). An increase of 15% inthe respiration rate of hand-peeled carrots was detected when compared to unpeeledcarrots. In contrast, abrasion peeling, which is more destructive than hand peeling,led to almost doubled respiration rates. For stored carrots, respiration rates increasedtwo- and threefold when fine-abrasion vs. coarse-abrasion peeling were used, respec-tively, in comparison with the rates observed for hand-peeled carrots. Shredded iceberglettuce had a 35–40% increase in respiration rate in relation to quartered lettuceheads. The type of equipment used may also affect the physiological response ofthe tissues—sharp rotating blades gave better results in cutting lettuce (lower respi-ration and lower microbial count during storage) than sharp stationary blades(O’Beirne, 1995). Evidently, the tissue response to mechanical injury is expected tobe more pronounced when extensive wounding is inflicted on the produce, such as

TABLE 9.3Relative PPO Activity in Different Apple Cultivars

Cultivar

Relative PPO Activity

Peel Cortex

‘Red Delicious’ 100 100‘Golden Delicious’ 33 30‘McIntosh’ 46 80‘Fuji’ 57 71‘Gala’ 30 48‘Granny Smith’ 43 73‘Jonagold’ 43 43‘Elstar’ 10 20

Source:

Adapted from Janovitz-Klapp et al., 1989.

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the grating of carrots vs. preparation of carrot sticks. Moreover, the direction of thecut also affects the tissue response to wounding (Zhou et al., 1992).

As a result of cutting, there is accumulation of cell fluids on the cut surface,and, in general, washing of cut produce may be helpful. Removal of cellular fluids(which carry potentially deleterious enzymes such as PPO, peroxidase, etc.) releasedduring the cutting operation is important and can be accomplished by simple rinsingprocedures. In addition, such fluids are nutrient-rich and facilitate microbial growth.Although washed mushrooms had 15% less soluble phenolics and showed leachingof PPO (two out of four isoforms) and, therefore, less enzymatic activity, there wasalso water uptake during washing. Consequently, a more rapid deterioration ofmushrooms, due to microbial spoilage and mechanical damage (Choi and Sapers,1994), was shown. Other commodities, such as lettuce, do not benefit from rinsing.Rinsed and drained shredded lettuce may retain 0.5–1% water on the surface, aresidual amount that can decrease product quality by facilitating decay; thus, de-watering has to be carried out (Bolin et al., 1977). However, centrifugation of lettuceto remove residual cold water may require spin conditions (speed, time) that resultin mechanical damage of the produce.

For many fruits and vegetables utilized by the fresh-cut industry, processing iscarried out shortly after harvest, but in some instances, the seasonality of harvestingmay not allow for this. Potatoes are an example of a vegetable that can be storedbefore use in the preparation of pre-peeled products. A Finnish study evaluated thetendency to brown of three potato varieties stored for different periods (Table 9.2).Results showed that only one variety (‘Bintje’) stored for one month would pass therequirements of the local industry, which establishes a maximum browning indexof 10 as acceptable for fresh-cut processing (Mattila et al., 1993).

Browning and Enzymes Other Than Polyphenoloxidase

Mechanical injury (wounding) and ethylene can stimulate phenolic metabolism infresh-cut tissue. Wounding and ethylene induce the activity of the enzyme pheny-lalanine ammonia lyase (PAL), a key enzyme for phenolic biosynthesis. Accumu-lated phenolic compounds can be used as substrates by PPO, leading to browning.It has been suggested that lettuce storage life is related to the activity of stress-induced PAL (Couture et al., 1993; Saltveit, 1997). In fresh-cut lettuce, browningof pieces is also a major detriment to quality. Different types of browning defectscan be observed in lettuce, such as russet spotting (RS) (which is characterized bybrown spots on the lettuce midribs), browning of cut leaf edges (LEB) and browningof the leaf surface (LSB). In wounded air-stored lettuce pieces, the major defectsdescribed are EB and LSB, while RS is most apparent in wounded ethylene-storedsamples. A comparison of the response of five types of lettuce (iceberg, romaine,green leaf, red leaf and butterhead) revealed differences in the maximum level ofwound-induced PAL, which were also affected by the storage of the whole lettuceheads before processing. Maximum levels of PAL decreased with increased storagetime (López-Gálvez et al., 1996). In addition, in harvested lettuce heads, the stemtissue near the harvesting cut may develop browning or so-called butt discoloration,when the cut stem initially becomes yellow, later develops a reddish-brown color, and

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finally develops an intense brown pigmentation. PAL activity is induced by cutting thelettuce stem, with subsequent synthesis and accumulation of soluble phenolic com-pounds (mainly caffeic acid derivatives), supplying substrates for PPO (Tomás-Barberánet al., 1997). PAL activity is believed to be proportional to the extent of wounding.

Peroxidase is an enzyme widely distributed in plants. Changes in peroxidase maybe brought about by wounding, physiological stress and infections. Many reactionscan be promoted by peroxidase, and in the presence of small amounts of hydrogenperoxide, it can oxidize a number of naturally occurring phenolics. Mono- and diphenolsare potential substrates for peroxidase (Robinson, 1991). It is believed that althoughperoxidase may also contribute to enzymatic browning, its role remains questionable(Nicolas et al., 1993) and limited by hydrogen peroxide availability (Amiot et al., 1997).

C

ONTROL

OF

E

NZYMATIC

B

ROWNING

Enzymatic browning may be controlled through the use of physical and chemicalmethods, and, in most cases, both are employed. Physical methods may includereduction of temperature and/or oxygen, use of modified atmosphere packaging oredible coatings or treatment with gamma irradiation or high pressure. Chemicalmethods utilize compounds that act to inhibit the enzyme, remove its substrates(oxygen and phenolics) or function as preferred substrate. Chemical means of con-trolling browning will be discussed first.

Prior to having their GRAS status revoked by the FDA in 1986 due to potentialhealth risks posed to sensitive consumers (Taylor, 1993), sulfites had a widespreadapplication in controlling both enzymatic and nonenzymatic browning. Followingtheir ban for use in fruits and vegetables to be consumed raw, other chemicals havebeen sought for prevention of enzymatic browning. Regardless of the fact that manydifferent PPO inhibitors have been used in research (Vámos-Vigyázó, 1981; McEvilyet al., 1992; Iyengar and McEvily, 1992; Sapers, 1993), in this chapter, only inhibitorswith potential application for fresh-cut fruits and vegetables will be discussed. It isimportant to point out that some chemicals used in research may not meet the safetystandards and pose toxic risks, others may impart undesirable sensory effects tofoods and others have shown effectiveness only in fruit juices but not on cut surfaces.

Traditionally, conventional food processing achieves the prevention of browningthrough heat inactivation of PPO, as with blanching and cooking. Heat inactivationis an effective method of browning prevention, and PPO is considered an enzymeof low thermostability, although differences in heat stability are reported for differentcultivars and PPO isoforms (Zawistowski et al., 1991). Nevertheless, use of heatalso has the potential to cause destruction of some food quality attributes, such astexture and flavor, and to result in nutritional losses. It is considered that in fresh-cut products, if heat treatments are applied, they should be minimized and shouldnot cause a cessation of respiration. Rather than, or in addition to, the use of heat,the control of enzymatic browning is frequently achieved through the use of differenttypes of chemicals, generally referred to as antibrowning agents.

For an enzymatic browning reaction to occur, essential elements are required: thepresence of active PPO, oxygen and phenolic substrates. Browning prevention is pos-sible, at least temporarily, through elimination of substrates and/or enzyme inhibition.

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Antibrowning Agents

Several types of chemicals are used in the control of browning (Table 9.4). Some typesact directly as inhibitors of PPO, others render the medium inadequate for the devel-opment of the browning reaction, and still others react with the products of the PPOreaction before these can lead to the formation of dark pigments.

Acidulants

While the optimum pH for PPO has been reported as ranging from acid to neutral, inmost fruits and vegetables, optimum PPO activity is observed at pH 6.0–6.5, whilelittle activity is detected below pH 4.5 (Whitaker, 1994). It has also been reported thatirreversible inactivation of PPO can be achieved below pH 3.0 (Richardson and Hyslop,1985). Nevertheless, it has also been reported that apple PPO is quite tolerant to acidity,and at pH 3.0, it retains 40% of its maximum activity (Nicolas et al., 1994).

The use of chemicals that lower the product pH, or acidulants, finds widespreadapplication in the control of enzymatic browning. The most commonly used acidulantis citric acid. Acidulants are frequently used in combination with other types ofantibrowning agents, because it is difficult to achieve efficient browning inhibitionsolely through pH control. In addition, there are variations in the effect of differentacids on PPO; as an example, malic acid has been reported to be more efficient inpreventing apple juice browning than citric acid (Ponting, 1960).

Reducing Agents

This type of antibrowning agent causes chemical reduction of colorless

o

-quinonesresulting from the PPO reaction back to

o

-diphenols (Iyengar and McEvily, 1992).Reductants are irreversibly oxidized during the reaction, which means that the pro-tection they confer is only temporary, because they are consumed in the reaction.When all the reducing agent added is oxidized, the

o

-quinones from the PPO reactionmay undergo further oxidation reactions (not involving PPO) and finally rapidpolymerization leading to the formation of brown pigments (Figure 9.2). Due to theoxidative nature of enzymatic browning, reducing agents can also be applied in theprevention of discoloration.

Ascorbic acid is probably the most widely used antibrowning agent, and inaddition to its reducing properties, it also slightly lowers pH. Ascorbic acid reducesthe

o

-benzoquinones back to

o

-diphenols, and it also has a direct effect on PPO(Whitaker, 1994; Golan-Goldhirsh et al., 1992).

Thiol-containing compounds, such as cysteine, are also reducing agents thatinhibit enzymatic browning. However, for complete browning control, the amountof cysteine required (cysteine-to-phenol ratios above 1) is often incompatible withproduct taste (Richard-Forget et al., 1992).

Chelating Agents

By complexing copper from the PPO active site, chelating compounds, such asethylenediamine tetraacetic acid (EDTA) can inhibit PPO, which is a metalloenzymecontaining copper in the active site. Sporix is a powerful chelator, and also anacidulant. Browning prevention in apple juice and cut surfaces was obtained withcombinations of Sporix and ascorbic acid (Sapers et al., 1989).

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Complexing Agents

This category includes agents capable of entrapping or forming complexes with PPOsubstrates or reaction products. Examples of this category are cyclodextrins or cyclicnonreducing oligosaccharides of six or more

D

-glucose residues. In aqueous solution,the central cavity of cyclodextrins can form inclusion complexes with phenolics,consequently depleting PPO substrates.

β

-Cyclodextrin has the most appropriatecavity size for complexing phenolic compounds, but its water solubility is low (Billaudet al., 1995).

β

-Cyclodextrin was not effective in controlling browning of diced apples,presumably due to its low diffusion (Sapers and Hicks, 1989). Large variations in theinhibitory properties of cyclodextrins have been found with different phenols tested.

β

-Cyclodextrin binding strength varies with different phenols. In model systemscontaining a single phenolic compound,

β

-cyclodextrin always works as a PPO inhib-itor. When mixtures of phenolic compounds were tested, the results were variable,and the balance among the PPO substrates present can be modified, resulting in colorchanges after enzymatic oxidation catalyzed by PPO (Billaud et al., 1995).

Enzyme Inhibitors

One of the antibrowning agents with the most potential for application to fresh-cutproducts is 4-hexylresorcinol, a chemical that has been safely used in medications fora long time and has been granted FDA GRASS (generally regarded as safe) status foruse in the prevention of shrimp discoloration (melanosis), where it proved to be moreeffective than sulfite on a weight-to-weight basis (McEvily et al., 1992). Currently, itsuse on fruit and vegetable products has been delayed while awaiting FDA approval.The efficiency of 4-hexylresorcinol has been demonstrated in preliminary tests carriedout using cut apples and potatoes (McEvily et al., 1991). The combination of 4-hexylresorcinol with ascorbic acid improved browning control in apple slices (Luoand Barbosa-Canovas, 1995).

Other Antibrowning Agents

Sodium chloride (as other halides) is known to inhibit PPO; its inhibition increasesas pH decreases. Chloride is a weak inhibitor; some authors report that the chloridelevels required for PPO inhibition are elevated and may compromise product taste(Mayer and Harel, 1991). Nevertheless, other authors believe that browning controlmay be possible provided that the dipping solutions are acidic; a pH of at least 3.5has been suggested (Rouet-Mayer and Philippon, 1986).

Calcium treatments used for tissue firming have also been reported to reducebrowning (Drake and Spayd, 1983; Hopfinger et al., 1984; Bolin and Huxsoll, 1989).Although citric acid and/or ascorbic acid dips were not effective in preventingbrowning of pear, slices dipped in 1% CaCl

2

and stored for a week at 2.5

°

C appearedto be lighter in color than water-treated control slices (Rosen and Kader, 1989). Infact, this could be due to the PPO inhibition by the chloride ion (Table 9.4). Nevertheless,the firming action of calcium (pages 292–293) could contribute to a reduced leakageof PPO and its substrates at the exposed cut surfaces (Sapers and Miller, 1992).

Antibrowning activity has been attributed to a small peptide isolated from honey.Browning inhibition (62%) in slices of peeled apples has been achieved by dippingin a 10% honey solution for 30 minutes at room temperature. Comparison with a

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TABLE 9.4Chemical Agents with Inhibitory Action on Enzymatic Browning

Browning Inhibitor Effect/Action Shortcomings CommentsExamples of Tested

Concentrations

Acidulants

Citric acid Possible dual effect: lowering pH and chelating Cu from PPO active site

Frequently used in combination with other agents

Theoretically, inhibition of enzymatic browning can be achieved by lowering the pH 2 or more units below the PPO optimum pH

(1)

0.5–2% (w/v)

(2)

Other organic acids: Tartaric acid, malic acid, lactic acid

Lower pH Cost Limited availability

Inorganic acids: Phosphoric acid, hydrochloric acid

Lower pH Sensory effects: taste

Reductants

(

Reducing Agents; Antioxidants

)

Ascorbic acid (AA) Reduction of

o

-quinones to colorless diphenols

Temporary effect; ascorbic acid is also consumed

Nonspecific; can cause formation of off-colors and/or off-flavor

Insufficient penetration into the food tissues

0.5%–1% (AA, EA)

(3)

Erythorbic acid (EA) Same as ascorbic acid Some authors reported that erythorbic acid is destroyed at a faster rate than ascorbic acid

0.8%–1.6% (AA, EA)

(4)

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Ascorbyl-phosphate esters AA-2-phosphate (AAP) AA-triphosphate (AATP)

Release ascorbic acid upon hydrolysis by acid phosphatase present in plant tissues

Less effective than ascorbic acid in some applications, more effective in others (depends on the phosphatase activity of each tissue)

More stable to oxidation than ascorbic acid

45.4 mM (0.8% AA)

(5)

Sulfhydryl compounds:

L

-cysteineReact with

o

-quinones producing stable adducts (colorless)

Expensive Potential formation of off-flavors at the required concentration

More effective than ascorbic acid 230 mM

(6)

Complexing Agents

Cyclodextrins (cyclic oligosaccharides)

Formation of complexes with PPO substrates

Entrapment of PPO substrates or products

Complex formation is not specificPotential removal of color and/or flavor compounds

Cost: Not approved yet

Suggested use also in combination with other agents (acidulants, chelators, reductants)

(9)

β

-cyclodextrin

(

β

-

CD) Suggested use in juices Water solubleLower levels were required when combined with phosphates

(10)

1–4%

(2)

maltosyl-

β

-CDhydroxyethyl-

β

-CDMore soluble than

β

-CD 4%

(10)

10%

(10)

Chelating Agents

EDTA Metal chelator: Binds copper at the PPO active site and copper available in the tissue

Commonly used in combination with other antibrowning chemicals

Levels up to 500 ppm are permitted for disodium EDTA and calcium and disodium EDTA

Polyphosphate Chelators Low solubility in cold water Used in combination with other agents

0.5–2%

(2)

(

continued

)

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TABLE 9.4Chemical Agents with Inhibitory Action on Enzymatic Browning (Continued)

Browning Inhibitor Effect/Action Shortcomings CommentsExamples of Tested

Concentrations

Sporix

TM

(acidic polyphosphate)Chelator and acidulant Not approved in the U.S. for food use Although ineffective when tested

alone on apple plugs, in combination with ascorbic acid, it was very effective (apparent synergism)

0.24% Sporix + 1% ascorbic acid

(8)

Sodium acid pyrophosphate, Sodium Hexametaphosphate

Enzyme Inhibitors

4-hexyl resorcinol PPO inhibitor Not approved for use in fruits and vegetables

Specific action on PPOWater solubleChemically stableSafely used in the prevention of shrimp pigmentation (GRAS status)

Other potential food usesAnions: Chloride(NaCl, CaCl

2

)(ZnCl

2

)Interaction with copper at the PPO active site

Weak inhibitor at low to moderate concentration

Inhibition increases at lower pHs 2–4%

(2)

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Miscellaneous

Enzymatic treatment with proteases

Proteolysis Fig preparations revealed compounds other than ficin, resorcinol derivatives, which are PPO inhibitors

CostFicin (from fig), Bromelain (from pineapple), Papain (from papaya)

0.5% w/v solution

(11)

Honey Contains a small peptide that inhibits PPO

20% solution

(12)

Note:

References: (1) Whitaker and Lee, 1995; (2) McEvily et al., 1992; (3) Santerre et al., 1988; (4) Sapers and Ziolkowski, 1987; (5) Sapers et al., 1989; (6) Kahn, 1985;(7) Montgomery, 1983; (8) Gardner et al., 1991; (9) Hicks et al., 1990; (10) Hicks et al., 1996; (11) Labuza et al., 1992; (12) Oszmianski and Lee, 1990.

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control sucrose solution at the same sugar level as the honey preparation showedonly a 23% inhibition of browning (Oszmianski and Lee, 1990).

Enzymatic treatments with proteases that attack PPO have been suggested asalternative prevention treatments for enzymatic browning. It was presumed that PPOinhibition by proteases was due to proteolysis or to binding at specific sites requiredfor activation. Another possible mechanism of action suggested was related to thepresence of sulfhydryl groups (such as cysteine) in the proteases. Enzymatic treat-ment of PPO could potentially be carried out with bromelain (extracted from pine-apple), papain (from papaya) and ficin (from figs). Preliminary tests were done usingsmall pieces of apples and potatoes that were dipped for 5 minutes in a 2% enzymesolution in citrate buffer at pH 4.5. Results showed that papain worked best onapples, while ficin worked better on potatoes. Parallel tests on untreated samplesand control citrate buffer-dipped samples developed comparable discoloration(Labuza et al., 1992). However, partially purified ficin preparations, where theprotease was heat inactivated, were comparable to preparations containing activeficin as PPO inhibitors (McEvily, 1991). Later, it was found that ficin preparationscontain, in addition to the protease, other antibrowning agents that are analogs of4-substituted resorcinol (McEvily et al., 1992). Extracts prepared from papaya con-tain cysteine and another “quinone-trapping” substance identified as a dipeptidecysteine-glutamic acid (Richard-Forget et al., 1998).

Although benzoic and cinnamic acids (aromatic carboxylic acids) are PPOinhibitors (Walker, 1975), they have not given prolonged protection as antibrowningagents. When solutions of sodium cinnamate were used to dip apple plugs, browningprevention was obtained on a short term, but over prolonged storage (

>

24 hr), asevere browning developed (Sapers et al., 1989). It has been suggested that cinnama-tes and benzoates may undergo a slow but gradual conversion to PPO substrates(Sapers et al., 1989; McEvily et al.,1992).

There are consumers who want to avoid any type of food preservative (Bruhn,1995). It is recognized that the consumer perceives fresh-cut products as minimallyprocessed products with characteristics close to their raw unprocessed material.Flavor, color and texture characteristics are probably an added appeal of fresh-cutproducts, and as a consequence, some processors would rather not use chemicaladditives that could change that perception of a “natural” product. This may be oneof the reasons that ascorbic acid, which may be labeled as vitamin C, is frequentlypreferred as an antibrowning agent, an added value to the product. Other chemicalsof natural origin or identical to natural compounds are also frequently preferred, anexample of which is citric acid. With this in mind, some authors have tested theefficiency of other natural products, such as pineapple juice, in the control of enzymaticbrowning. Among the constituents of pineapple juice, antibrowning activity could beattributed to both ascorbic acid, but in addition, the juice contains a low molecularweight inhibitor which is as yet uncharacterized (Lozano-de-Gonzalez et al., 1993).

Application of Antibrowning Agents

In general, chemicals used to prevent or control enzymatic browning are used insolutions, frequently as formulations containing one or more compounds that areused for dipping the fruit or vegetable pieces. It has been reported that with some

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chemicals, such as ascorbic and erythorbic acid or their salts, limited penetrationinto the plant tissue is an issue. A comparison of the effect of dipping vs. pressureor vacuum infiltration on the penetration of ascorbic and erythorbic acids showedthat pressure infiltration was ineffective with potato dice but extended the shelf lifeof potato plugs by two to four days when compared to dipping (Sapers et al., 1990).The variation in response of potato plugs and dice to pressure infiltration wasattributed to the smaller surface-to-volume ratio in the plugs. The authors of thestudy suggested that the technique could be applied to larger pieces, even peeledtubers. With apple plugs and dice, the pressure infiltration method was superior todipping, providing an increase of three to seven days in the storage life of applepieces. Nevertheless, infiltrated dice can become waterlogged and require dewateringby centrifugation or partial dehydration to overcome that defect. In addition, if toomuch pressure is applied, cell rupture can occur leading to loss of textural integrityand perhaps reduced shelf life.

Combined Treatments

More effective preservation of fresh-cut products can frequently be achieved byusing a combination of treatments. A common treatment combination includesascorbic acid and calcium chloride, such as presented in Table 9.5 (Ponting et al.,1972). In the case of two apple varieties, e.g., ‘Newton Pippin’ and ‘Golden Deli-cious,’ the highest concentrations of ascorbic acid (1%) and CaCl

2

(0.1%) utilizedresulted in the lowest loss of reflectance or browning readings. It is interesting thatthe use of CaCl

2

alone caused almost as much inhibition on ‘Newton Pippin’ apples,but this was not so for ‘Golden Delicious.’ Table 9.6 shows some results from a

TABLE 9.5Effect of Treatments with Ascorbic Acid (AA) and Calcium Chloride on the Prevention of Discoloration in Apple Slices

Treatment

1

Loss of Reflectance (%) Compared to Freshly

Sliced Apples

var. ‘Newton Pippin’ var. ‘Golden Delicious’

Control—water dip 62.5 60.50.05% CaCl

2

24.8 58.90.1% CaCl

2

23.3 51.20.5% AA 57.9 59.20.5% AA

+

0.05% CaCl

2

26.9 48.00.5% AA

+ 0.1% CaCl2 24.2 25.61% AA 25.5 45.61% AA + 0.05% CaCl2 20.5 39.21% AA + 0.1% CaCl2 4.2 17.0

1Three-minute dip in 1 L of antibrowning solution, followed by 1 min drainingand packaging in plastic bags prior to storage at ~1°C for 11 weeks.

Source: Adapted from Ponting et al., 1972.

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TABLE 9.6Effect of Combined Treatments on the Browning Index of Potato Slices 2 Hours After Cutting

Antibrowning Agent pH

Browning Index

var. ‘Bintje’ var. ‘Van Gogh’ var. ‘Nicola’

1 mo. 5 mo. 8 mo. 1 mo. 5 mo. 8 mo. 1 mo. 5 mo. 8 mo.0.3% AA + 0.5% citric acid 2.4 0 1 1 0 6 2 4 3 30.5% AA + 0.5% citric acid 2.4 0 2 0 1 3 2 6 1 20.3% AA + 0.3% citric acid + 0.1% CaCl2 2.4 0 2 2 6 5 2 4 2 40.3% AA + 0.3% citric acid + 0.2% K sorbate 3.2 0 3 3 4 4 4 9 3 30.5% AA + 0.5% citric acid + 0.2% K sorbate 2.8 0 2 1 1 2 2 6 2 20.1% AA + 0.1% citric acid + 0.1% Na benzoate 3.5 0 4 2 2 7 3 4 8 60.5% citric acid + 0.005% 4-hexylresorcinol 2.6 0 1 2 0 3 2 5 3 3Water 5.7 1 4 6 10 22 9 67 39 13

Note: Dipping solution was applied at 5°C for 1 min in a ratio of 2 L of solution/kg of potato slices; slices were drained for 1 min and then kept for 2 h at 23°Cprior to browning evaluation.

Source: Adapted from Mattila et al., 1993.

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study using different combinations of antibrowning agents on slices prepared fromthree different potato varieties stored varying lengths of time (Mattila et al., 1993).Other combination treatments may include the use of antibrowning agents andphysical methods, such as a heat treatment or controlled atmosphere, such as thecombination of 0.5% O2 and 1% CaCl2, which was effective in minimizing browningin sliced pears (Rosen and Kader, 1989). In the preparation of pre-peeled potatoes,the damage inflicted by the peeling method has a significant effect on productdiscoloration. Unstable tissue from peeled potatoes can be removed by lye digestionor hot ascorbic acid/citric acid solutions prior to the treatment with browning inhib-itors (Sapers et al., 1995).

Physical Treatments and Browning Control

One of the most commonly used approaches to controlling enzymatic activity infresh-cut products is the use of low temperatures during handling, processing andstorage. At low temperatures, not only is enzymatic activity reduced, but generalmetabolic rates are also lower, which assists in extending product shelf life.

Some of the physical methods suggested for application in postharvest handlingof fruits and vegetables have also been proposed for fresh-cut products. These includethe use of modified/controlled atmospheres and gamma irradiation. Nonthermal meth-ods currently being investigated by food processors that may have application forfresh-cut products include high-pressure treatments or treatment with high electricfield pulses (Ohlsson, 1994).

Reducing Oxygen AvailabilityIt is important to consider that as a requirement of living tissues, fresh-cut productscannot be exposed to environments with complete removal of oxygen. Nevertheless,enzymatic browning can be delayed (in the presence of active enzyme and phenolicsubstrates) if oxygen is not available for the reaction to take place. In fruits andvegetables used for either conventional or fresh-cut processing, it is a commonpractice to hold preprepared produce (already peeled, cut, etc.) immersed in water,brine or syrup to retard diffusion of oxygen. However, tissue will brown when it isre-exposed to air. In addition, during the time the tissue is held, osmotic equilibriummay result in loss of solutes and imbibition of the storage solution.

Modified atmospheres are frequently used in packaging and/or storage of fruitsand vegetables. These conditions as well as edible coatings can also be successfullyadapted to fresh-cut fruits and vegetables (see Chapter 10).

Among other benefits, the use of modified or controlled atmospheres retardssenescence and, consequently, extends the storage life of products. Modified or con-trolled atmospheres should be seen as a supplements to adequate management oftemperature and controlled humidity (Kader, 1992).

Modified atmosphere packaging aims at the creation of an ideal gas compositionin the package that can be achieved through commodity-generated modified atmo-sphere in the package and through the establishment of an active modified atmospherein the package. However, it is important to avoid damaging low levels of oxygen orhigh levels of carbon dioxide which lead to anaerobic respiration, resulting in the

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development of off-flavors and off-odors and increasing the susceptibility to decay.Appropriate gas compositions, of modified atmosphere need to be experimentallydetermined for each particular product (Wills et al., 1998). Using a moderate vacuumpackaging with polyethylene (80 µm) for the storage of shredded iceberg lettuce at5°C, browning was inhibited over a 10-day period (Heimdal et al., 1995). Browningof commercially prepared cut lettuce was retarded in packaged product, where theatmosphere was altered by the respiring product. Visual quality of the cut lettuce pack-aged in sealed bags received an original score of 9 (excellent), and after storage fortwo weeks at 2.8°C, the score dropped to 7 (good). Samples stored in an unsealedpackage received a score of 3 (poor). Modified atmosphere packaging was alsoefficient in controlling microbial buildup during storage (King et al., 1991).

Shelf-life extension has also been investigated by enrobing fresh-cut productsin edible coatings. Such thin layers of protective materials are applied to the surfaceof the fruit or vegetable as a replacement for the natural protective tissue (epidermis,peel). Edible coatings are used as a semipermeable barrier that helps reduce respi-ration, retard water loss and color changes, improve texture and mechanical integrity,improve handling characteristics, help retain volative flavor compounds and reducemicrobial growth. It is possible to create a modified atmosphere enrobing fresh-cutproduce in edible coating (Baldwin et al., 1995a; Baldwin et al., 1996). Detailedinformation on edible coatings is presented in several reviews (Krochta et al., 1994;Baldwin et al., 1995a,b; Nisperos and Baldwin, 1996).

Basically, edible coatings are comprised of one or more major components (polysac-charides, proteins, resins, waxes or oils), which may be improved by the addition ofplasticizers, surfactants and emulsifiers. Appropriate selection of edible coatings isimportant due to the hydrophilic nature of cut surfaces of many fresh-cut products.Some coatings may not adhere to such surfaces, others may offer good adherence butmay be poor barriers to moisture or not resist water vapor diffusion (Baldwin et al.,1995a,b). Lipid components confer important water barrier characteristics to somecoatings, however, they may present a drawback, because they may give a waxy orgummy mouth feel to the product (Wong et al., 1994). On the other hand, hydrophilicpolymers (such as carboxymethyl cellulose) do not work well in reducing water lossof coated products, due to their poor moisture barrier characteristics (Baldwin et al.,1996). Emulsion coatings containing mixed components seem to have better perfor-mance, such as coatings of casein and acetylated monoglyceride; when the pH isadequately adjusted, a tight matrix is formed, trapping the lipid molecules (Krochtaet al., 1994). In addition, some lipid components (such as acetylated monoglyceride)are solid at room temperature, and without an emulsifier (such as calcium caseinate),they could not be used as a coating for fresh fruits and vegetables (Avena-Bustilloset al., 1997). In the application of some coatings, it is possible to induce the formationof cross-links between pectin molecules of the fresh-cut product surface and thecoating (Wong et al., 1994). Interestingly, different food additives can be incorpo-rated into coating formulations, such as coatings with antioxidants (Baldwin et al.,1995a). The efficiency of ascorbic acid in delaying enzymatic browning in cut appleand potato was improved when incorporated in an edible coating formulation incomparison to dipping. A carboxymethylcellulose-based coating did not controlenzymatic browning of cut apples and potatoes, but when such a coating was combined

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with additives (antioxidant, acidulant and preservative), browning control was supe-rior than dipping the fresh-cut produce in solutions with the same additives (Baldwinet al., 1996). Examples of browning inhibition of apple slices have been describedwith different edible coatings, such as formulations containing casein and lipid(Avena-Bustillos and Krochta, 1993) or soybean protein (Kinzel, 1992).

Reducing Temperature Temperature management during handling is essential in minimizing the damagingeffects of mechanical injury because of the ability of low temperatures to reducemetabolic reactions. Temperature has a tremendous effect on respiration rates; more-over, it affects permeability of gases through the packaging films and also slowsmicrobial growth. Fresh-cut products generally have higher respiration rates thanthe same intact produce—the respiration increase may vary from a few percent toover 100%. Moreover, the degree of respiration increase varies with temperatureand commodity (Watada et al., 1996). Storage temperature is a critical parameter inachieving maximum shelf life of products. Refrigeration throughout the productionchain to consumption is of fundamental importance in extending the shelf life offresh-cut products. To ensure high-quality products, it is recommended that fresh-cut products be kept at temperatures just above freezing; nevertheless, temperatureneeds to be adequately chosen in order to avoid damage such as chilling injury insensitive commodities. A common practice in the preparation of fresh-cut productsis rinsing the peeled and/or cut product in cold water, which helps lower the tem-perature in addition to removing cellular exudates released during the peeling and/orcutting of produce. Dewatering of rinsed products is normally required to controldecay. This is done commercially through centrifugation but can also be achievedwith forced air.

Although emphasis is normally placed on the use of low temperatures, there areexamples of benefits of some heat treatments on browning control. Heat shocktreatment (45°C for 105 min) of whole apples later used for preparing slices resultedin product with less browning and firmer texture than product prepared from non-heated fruit (Kim et al., 1993). In conventional food processing, the most widelyused methods for enzyme inactivation rely on heat application. Optimum PPO activityhas been reported to vary with the source of the enzyme and reaction conditions(pH, substrate, etc.). PPO from several plant sources exhibits maximum activity inthe temperature range of 20–35°C. Many factors affect PPO heat stability, among themare enzyme source, plant cultivar, molecular form (isozyme) and heat penetrationinto the tissue (Vámos-Vigyázó, 1981). PPO is not a very heat-stable enzyme; thermalinactivation occurs at temperatures higher than 40°C. Temperature stability of PPOdepends on the source of the enzyme. Moreover, PPO thermostability is also influ-enced by cultivar, growing location and pH (Vámos-Vigyázó, 1981; Nicolas et al.,1994). Banana PPO is inactivated in 15 min at 80°C (Galeazzi and Sgarbieri, 1978),while green pea PPO required 29 min at 80°C or 2.5 min at 90°C and only 1 minat 95°C (Krotov et al., 1971). Low-temperature blanching may be effective in pre-venting or controlling enzymatic activity in fresh-cut products. Blanching (95°C for3 min) of ready-to-use pear cubes under aseptic conditions resulted in completeinhibition of enzymatic browning with an acceptable texture reduction, as judged

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by a trained panel (Pittia et al., 1999). Recently, heat shock treatment has beensuggested as a new way to control browning in fresh-cut products. The mechanicalinjury caused by tissue wounding induces synthesis of enzymes, such as phenyla-lanine ammonia lyase (PAL), involved in phenolic metabolism, leading to accumu-lation of phenolic compounds, which in turn can be potential substrates for PPO.Within 24 hours of cutting, iceberg lettuce cut into 2 × 2 cm pieces showed a six-to 12-fold increase in PAL activity. A heat shock treatment on cut iceberg lettucefor 90 seconds at 45°C prevented such increase in PAL activity, which might offera new alternative to control browning in fresh-cut products (Saltveit, 2000).

Applying Gamma RadiationApplication of gamma radiation to fruits and vegetables has been used for insectand disease disinfestation, as well as to retard ripening and sprouting. Irradiationapplied to fresh-cut carrots stored in microporous plastic bags resulted in limitedrespiration increase due to wounding and reduced ethylene production. Treatment wasconsidered to increase the shelf life of the product (Chervin et al., 1992). Neverthe-less, the application of irradiation may bring about undesirable biochemical changes.In fact, enzymatic browning may be aggravated by irradiation treatments, whichmay alter the permeability of cell compartments favoring contact between PPO andits substrates (Mayer and Harel, 1991). Apples and pears irradiated as a quarantinetreatment showed decreased firmness, which was cultivar dependent, and changesin internal color of ‘Gala’ and ‘Granny Smith’ apples (Drake et al., 1999). Endivesamples that were irradiated revealed longitudinal internal pink-brown lines, whichprogressed to the entire vegetable piece becoming pink-brown. In contrast, the cutcontrol discolored only on cut surfaces (Hanotel et al., 1995). Such alterations maybe an indication of cell damage, release of PPO and browning in the irradiated endive.

Use of Other Nonthermal TechnologiesHigh-pressure processing has applications in food preservation due to its potentialeffect on microorganisms and enzymes. Inactivation of deleterious enzymes has beenachieved through application of high-pressure technology (Hendrickx et al., 1998;Seyderhelm et al., 1996; Weemaes et al., 1999). An important advantage of this newtechnology is that high-pressure treatments at low temperatures have either no effector a minimal effect on flavor and nutritional value of foods. However, high-pressureprocessing may create new textures or tastes (Messens et al., 1997), and cause discol-oration of some commodities (Asaka and Hayashi, 1991).

While bacterial spores are highly resistant to pressure treatment, and over 1200MPa is required for their inactivation, yeasts, molds and vegetative cells are pressuresensitive and can be inactivated by milder treatments at ~300–600 MPa. When aimingat enzyme inactivation, pressure requirements vary depending on the enzyme; someenzymes are resistant to 1000 MPa, others can be inactivated by a few hundred MPaat room temperature. High pressure has been considered an alternative for irreversibleinactivation of PPO (Hendrickx et al., 1998). It has been observed that the applicationof low pressure results in pressure-induced membrane damage with consequentdecompartmentalization and enzyme activation. In fact, pear PPO (cell-free extracts)was activated after pressure treatment at 400 MPa for 10 min at 25°C (Asaka and

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Hayashi, 1991). PPO activation was also described in low-pressure treatments ofcrude carrot and apple extracts (Anese et al., 1995).

PPO sensitivity to pressure varies with the enzyme source—while apricot PPOhas been inactivated at ~100 MPa, and strawberry PPO at 400 MPa, potato andmushroom PPO required much higher pressures (~800–900 MPa). In addition, PPOinactivation by pressure is affected by pH (Anese et al., 1995).

Many studies have been carried out in model systems, cell-free or crude extracts,not real foods. Experiments carried out on whole foods revealed that high-pressuretreatment caused browning of mushrooms, apples and potatoes (Gomes and Ledward,1996). It is known that food ingredients have a protective effect on enzyme pressurestability, and the efficiency of the pressure treatment depends on pH, temperatureand treatment. When comparing the barostability of different food enzymes, PPOwas second, only after peroxidase, as the most tolerant to pressure treatment duration(Seyderhelm et al., 1996). Due to the poor effectiveness of lower pressure treatmentson PPO activity, it has been suggested that its inhibition would require a combinationof pressure treatments with one or more additional methods, such as blanching,modified atmospheres and/or refrigeration (Anese et al., 1995). Complete inactiva-tion of enzymes is not expected with the application of hydrostatic pressures com-patible with maintenance of food tissue integrity (Whitaker, 1996).

The use of high-intensity pulsed electric fields is a new technology that has beensuggested to inactivate microorganisms and enzymes with minimal resultant tem-perature increase (Qin et al., 1996). Application of high-intensity electric field pulseson a culture of potato cells increased the release of PPO into the medium, with bothincreased intensity and duration of treatment (Knorr and Angersbach, 1998). Pre-liminary results using model systems (enzyme solutions) resulted in large variationsamong enzymes. Although a reduction of 88% in pectinesterase activity has beenreported in treated orange juice (Hye et al., 2000), a moderate activity reduction of30–40% was described for PPO and peroxidase treated in buffer solutions (Ho et al.,1997). Although this technology seems to offer potential applications to liquid foods,it still seems premature to recommend its use in fresh-cut products.

OTHER COLOR CHANGES

White Blush in Carrots

The bright orange color of fresh carrots can disappear in stored fresh-cut products,particularly when abrasion peeling is used. Carrots may develop “white blush,” alsoknown as “white bloom,” a discoloration defect that results in the formation of awhite layer of material on the surface of peeled carrots, giving a poor appearanceto the product. Upon peeling, the protective superficial layer (epidermis) of carrotsis removed, generally by abrasion, leaving cell debris and an irregular surface, whichwhile moist, presents the natural orange color of carrots. Once the carrots are exposedto air, they easily dehydrate, and the dried cell debris acquires a whitish color, forminga white layer on the carrot surface. The disruption of surface tissues followed bydehydration in white blush formation was confirmed by scanning electron microscopy,when comparing carrots peeled with a knife and a razor-sharp blade. Knife-peeled

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carrot surfaces appeared severely damaged, compressed, sloughed and separatedfrom underlying tissue, and therefore, prone to dehydration. Razor-peeled carrotsurfaces were cleaner, and apparently, only a thin layer of cells had been removed,resulting in a product that upon drying did not acquire the whitish appearance(Tatsumi et al., 1991). At this stage, the quality defect can be reversed by dippingthe carrots in water and allowing for rehydration (Cisneros-Zevallos et al., 1995).

It has been suggested that with time, phenolic metabolism may be activated,inducing increases in lignin, phenolic compounds and phenylalanine ammonia lyaseactivity, and irreversible color change takes place (Cisneros-Zevallos et al., 1995;Howard and Griffin, 1993). A positive test for lignin was described in the whiteabraded material. The severity of the lignification will depend on the harshness ofthe peeling process (coarse sandpaper > fine sandpaper > stainless steel pad).Although hand peeling of carrots with a razor blade has been reported to result inno development of “white blush,” even after prolonged storage at 1°C (Bolin andHuxsoll, 1991), the extent of injury of fresh-cut carrot discs depends on the bladetype and sharpness, and storage conditions. Carrot discs prepared by hand slicingwith a razor blade resulted in better quality products as compared to mechanicalslicing. Great damage was inflicted by a blunt machine blade; after 10 days of storageat 8°C, carrot discs sliced with a blunt machine blade revealed thickened cell wallsand the presence of lignin (Barry-Ryan et al., 2000). As the lignification process isenzyme mediated, some dipping treatments directed to inactivate the responsibleenzymes have been tested. A successful result was obtained with a treatment com-bining heat inactivation and an acidic environment. Carrots peeled with coarsesandpaper and dipped for 20–30 sec in a 2% citric acid solution at 70°C did notdevelop the defect for at least five weeks in cold storage; product taste was not affectedby the treatment (Bolin and Huxsoll, 1991). Edible films have also been shown toprotect carrots from this quality defect (Sargent et al., 1994). Sensory results showedpreference for carrots coated with an edible cellulose-based coating due to a freshappearance (Howard and Dewi, 1995), because consumers perceive white blushcarrots as not fresh or aged. Losses of carotenes have been described in fresh-cutcarrots. With the application of an edible coating, a 50% retention of β-carotene wasobtained after 28 days of storage, compared to 33% retention in the control (Li andBarth, 1998). Edible coating emulsions containing caseinate-stearic acid were effec-tive in reducing the white blush defect of carrots (Avena-Bustillos et al., 1994).

Yellowing or Degreening

Reduction of green pigmentation and, therefore, the predominance of yellow pigmentsis a normal process in ripening or senescence of many fruits and vegetables, and suchchanges can be accelerated by ethylene. In fresh-cut products the stress imparted bywounding results in increased respiration, ethylene production and other alterations.In fact, degreening is also observed during storage of leafy and other green fresh-cut products. Shredded iceberg lettuce darkens during storage, particularly at hightemperatures. Simultaneously, loss of green pigmentation was observed (Bolin et al.,1977; Bolin and Huxsoll, 1991). Studying the susceptibility of fresh-cut baby andromaine lettuces to browning, it was observed that samples of photosynthetic tissue

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became lighter during storage. In fact, while there is mid-rib discoloration, the pho-tosynthetic tissues also develop browning and loss of green pigments (Castañer et al.,1999). In a study on coleslaw color, over a period of cold storage, changes werefrom green to a lighter white color, suggesting chlorophyll degradation resulting incolorless compounds (Heaton et al., 1996). The reactions involved in the loss ofchlorophyll in green fresh-cut products are still unclear. During the preparation stepsof fresh-cut products, acids and enzymes are released, and both could be involvedin the loss of green pigmentation.

The visual quality of broccoli is lost when florets turn yellow. The retention ofgreen color has been attained with the use of modified atmosphere packaging andstorage at 10°C (Barth et al., 1993). These authors found that within 48 hours, thecarbon dioxide concentration inside broccoli packages reached equilibrium at ~8%and oxygen content was 10%, causing a reduction of respiration rate. Modifiedatmosphere packaging contributed to significantly higher retention of green color inbroccoli, as indicated by the total chlorophyll levels and color determination (hueangle). In contrast, in less than 72 hours, nonpackaged samples lost about 20% ofinitial chlorophyll content. Although there was ethylene accumulation during stor-age, it is suggested that the elevated carbon dioxide atmosphere counteracted eth-ylene effects, thus preventing chlorophyll degradation. Furthermore, the packagingof broccoli spears resulted in improved retention of vitamin C.

In a study of texture improvement, calcium-chloride-treated fresh-cut green pepperand nontreated control stored at 10°C had significant losses in green color after fourdays of storage. Calcium-treated samples stored at 5°C were significantly better inall sensory attributes by day four, and their superiority was maintained throughoutthe eight-day storage period (Barrett, unpublished).

PREVENTION OF TEXTURE LOSS IN FRESH-CUT PRODUCTS

Appearance of a food product plays an important role in consumer evaluation. Ithas been estimated that 95% of American consumers take appearance into accountin their purchases of fruits and vegetables (Shewfelt, 1994). As mentioned earlier,color has a great impact on appearance consideration, but quality loss is also observedwith changes in texture (Figure 9.1), another important quality criteria for manyfruit and vegetable products.

While genetic background is the major contributor to the texture of a plant food,other factors, such as morphology, cell wall-middle lamella structure, cell turgor,water content and biochemical components, all affect texture (Harker et al., 1997).In addition, texture is also affected by growing conditions, including environmentalfactors and production practices (Sams, 1999). After harvesting, it is important tostore fruits and vegetables at the appropriate temperature and relative humidity topreserve their quality. Storage temperature has a major effect on water, weight lossand metabolic activity.

In general, perishability of intact fruits and vegetables correlates well with res-piration rates—produce with a high respiration rate tends to be more perishable. Infresh-cut products, as a result of wounding, respiration is elevated compared to the

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intact produce. Moreover, the extent of wounding also affects the shelf life ofproducts. Hand tearing of lettuce has been shown to be less damaging than slicingwith rotating knives, and reduction of lettuce piece size shortens shelf life (Bolinand Huxsoll, 1991).

FRUIT AND VEGETABLE TISSUE FIRMING

During fruit ripening, one of the most notable changes is softening, which is relatedto biochemical alterations at the cell wall, middle lamella and membrane levels.Although pectic enzymes, polygalacturonase and pectin methylesterase have beenattributed to a significant role in the softening process, the precise mechanism isstill unclear.

Calcium and/or Heat Treatments

It is well known that calcium is involved in maintaining the textural quality ofproduce. Calcium ions form cross-links or bridges between free carboxyl groups ofthe pectin chains, resulting in strengthening of the cell wall. A common treatmentused to improve tissue firmness is to dip fruit or vegetable pieces in calcium solutions,as described for strawberries (Main et al., 1986), pears (Rosen and Kader, 1989)and shredded carrots (Izumi and Watada, 1994), among others. In contrast, calciumtreatment was not effective in carrot slices and sticks, a fact attributed to insufficientcalcium absorption by the tissue, because the levels of calcium were two and threetimes higher in shredded carrots than in sticks and slices, respectively. In addition,increasing the concentration of CaCl2 in the dip solution (0.5% or 1%) brought anincrease in the tissue calcium content of treated samples, without a subsequentcorrelation with product texture (Izumi and Watada, 1994).

A combined treatment associating low-temperature blanching to activate theenzyme pectinesterase (PE) prior to the calcium dip is helpful in preserving fruittexture. PE brings about the de-esterification of pectin, thus increasing the numberof calcium-binding sites. To such mechanism has been attributed the firming effectobserved in apple slices kept at 38°C for six days immediately after harvest, andsliced and dipped in calcium solution after six months of cold storage (Lidster et al.,1979). In fresh-cut melon cylinders dipped in calcium chloride solutions at differenttemperatures (Luna-Guzmán et al., 1999), texture was firmer in samples treated at60°C (77% improvement in firmness) than at 40°C (58% improvement) and 20°C(45% improvement).

Frequently, calcium chloride has been used as a firming agent, however, it mayconfer undesirable bitterness to the product. Fresh-cut cantaloupe cylinders dippedin calcium lactate solutions resulted in a textural improvement similar to calcium-chloride-treated fruit cylinders. Sensory evaluation indicated that results were better,e.g., less bitterness and a more detectable melon flavor was perceived. Fresh-cutcantaloupe cylinders treated by a combination of heat treatment (60°C) and calciumlactate dip were not significantly different either in bitterness or firmnes in relationto fruit treated at 25°C (Luna-Guzmán and Barrett, 2000).

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Heat treatment alone has been shown to have the potential to benefit producttexture. In a comparison of 11 apple cultivars, heat treatment of whole fruit resultedin firmer products when compared with nonheated fruit. The best firmness improve-ment was obtained with ‘Golden Delicious’ and ‘Delicious’ apples (Kim et al.,1993). Heat treatment of whole apples improved apple slice firmness, but the storagetemperature of whole fruit after heating had a significant effect on product firmness,except for fruit of the cultivar ‘McIntosh.’ Slices prepared from previously heat-treated whole apples stored at 2°C were firmer than products from fruit kept at 10,18 and 25°C for seven days. Slices prepared from heat-treated apples showedincreased firmness during storage of up to seven days for ‘Golden Delicious’ (firm-ness 34% higher than on day zero of storage) and up to 14 days for ‘Delicious’apple (48% higher firmness than at the beginning of storage). With longer storagetimes, there was a decrease in firmness for both cultivars (Kim et al., 1994).

Gamma-irradiated apple slices showed firmness reduction dependent on theirradiation dose applied. Significant softening was observed at doses above 0.34kGy. Although the total pectin content was unaltered, there was an increase in thecontent of water-soluble pectin in the irradiated slices. Calcium treatment of thick( of an apple) fruit wedges prior to irradiation led to a small firmness improvementin comparison to the softening brought about by radiation at 1 kGy. The inefficiencyof calcium in preventing irradiation-induced softening could be due to the limitedpenetration of calcium into the treated cut apples. When thin (3–4 mm) apple ringswere treated with calcium chloride (2 to 4%) and then irradiated, softening wasreduced proportionally to the calcium levels, although firmness was still lower thannon-irradiated controls (Gunes et al., 2001).

Use of Modified Atmosphere Packaging

Controlled atmospheres retard senescence, lower respiration rates and slow the rateof tissue softening (Kader, 1992). Texture loss has been reported to decrease incontrolled atmosphere packaging of fruit. Strawberry slices kept under controlledatmosphere for a week had comparable firmness to whole and freshly sliced fruit(Rosen and Kader, 1989). The authors suggest that the effect of controlled atmo-sphere on firmness appeared to be cultivar dependent.

Storage stability of halved fruits was evaluated in a combination treatment thatincluded chemical dips to prevent browning and retard texture loss, complementedby different storage conditions. Table 9.7 presents the texture results for peach halvesstored in sealed packages where oxygen was being consumed with accumulation ofcarbon dioxide and sealed packages containing an oxygen scavenger; the lattertreatment gave better results. Interestingly, a peach cultivar with soft texture, ‘Suncrest,’even showed an increase (+0.5 N/wk) in fruit firmness during the first few weeksof storage with oxygen scavengers. Similar storage conditions were not successfulin the treatment of pears. The authors suggest as an optimum treatment for halvedpeaches and apricots a combination of a dip in 2% calcium chloride and 1% zincchloride, followed by packaging with an oxygen scavenger and storage at 0–2°C(Bolin and Huxsoll, 1989).

18---

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WATER LOSS PREVENTION

After harvest, produce must utilize internal moisture solely; water lost through tran-spiration cannot be replaced. Although plant tissues are mainly composed of water,even small changes in water content may have a large impact on produce quality,causing losses that may occur in a few hours under dry and warm conditions. Waterlosses of 3 and 5% in spinach and apple, respectively, render these commoditiesunmarketable (Sams, 1999). Crispness of fresh produce is related to turgor pressure,whose loss may also contribute to softening. Leafy vegetables are particularly sus-ceptible to desiccation because of their large surface-to-volume ratios; moreover,loose leaves, such as spinach, are more prone to desiccation than a compact head,such as a whole lettuce head (Salunkhe and Desai, 1984). As a consequence of waterloss, appearance changes such as wilting and reduced crispness may occur.

Fresh-cut products tend to be more vulnerable to water losses because they areno longer intact after peeling and cutting or shredding, slicing, etc. Peel or skin isa very important barrier to loss of turgor and desiccation. Many commodities havea protective waxy coating that is highly resistant to water loss. Evidently, peelremoval renders commodities more perishable. The mechanical injury brought onby cutting and the method used, directly expose the internal tissues to the atmosphere,promoting desiccation. The shredding or slicing operations result in increased surfacearea, an additional problem. Moreover, mechanical injury brings about physiologicalresponses, such as respiration increase and, potentially, ethylene production, responsesthat shorten the life of a commodity. When rinsing of products is done after cutting,this is frequently followed by centrifugation. If accelerated centrifugation speed orlong centrifugation times are applied, increased desiccation can result, as reportedfor cut lettuce (Bolin and Huxsoll, 1989). Appropriate handling techniques includingtemperature and relative humidity control can help minimize the rate of water loss.Reduction of water loss can be achieved basically by decreasing the capacity of thesurrounding air to hold water, which can be obtained by lowering the temperatureand/or increasing the relative humidity. To reduce the rate of water loss in coolstorage, it is also important to restrict the air movement around the commodities

TABLE 9.7Texture Loss in Fresh-cut Freestone Peach Halves Stored for Seven Weeks at 2°C

Peach Variety

Initial Texture Modified Atmosphere With O2 Scavenger

(N)Texture

(N)Rate of Loss (N/Week)

Texture (N)

Rate of Loss (N/Week)

‘Fairmont’ >21 8.0 1.7 17.0 0.5‘Suncrest’ 7 4.0 0.5 10.5 +0.5‘Flamecrest’ 21 3.9 2.4 7.6 1.9

Note: Texture measurement units in Newtons (N).

Source: From Bolin, H.R. and Huxsoll, C.C. (1989). J. Food Process. Preserv. 13:281–292. With permission.

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(Wills et al., 1998). The primary parameter affecting celery quality is water loss,small, reductions in moisture (2.5–5%) may lead to flaccidity, shriveling, wrinklingand pithiness. Significant increases in moisture retention by celery sticks weredescribed with the application of a caseinate-acetylated monoglyceride coating(Avena-Bustillos et al., 1997). Additionally, it is important to point out that appro-priate packaging is of enormous importance in preserving fresh-cut products.

APPENDIX: EVALUATION OF ENZYMATIC BROWNING

Different authors have used somewhat different methods to measure the intensity ofenzymatic browning, and some tried to establish correlations of browning with PPOactivity and/or phenolic substrates content. Frequently, browning evaluation is basedon reflectance measurements on exposed surfaces, such as cut fruits and vegetablesor produce homogenates. Laboratory assays commonly involve extraction of brown-ing products and measurement of absorbance at particular wavelengths. Neverthe-less, as not all PPO products are soluble, some authors have developed methods thatalso evaluate the insoluble colored products.

Following, we present a brief description of some of the methods that were usedby authors whose results are displayed in tables included in this chapter. A morecomplete approach for assessing susceptibility to browning is also included, and atlast, we present a visual assay that can be used in assessing produce varieties tendencyto discoloration.

REFLECTANCE MEASUREMENTS

Table 9.1 presents results in DL* (decrease in L* indicates darkening of the samples)(Radi et al., 1997).

Homogenates (purees) of previously dried apricots were poured in small petridishes, and reflectance measurements (L* lightness; a* green/red; b* blue/yellowchromaticity) were taken with a Minolta CR300 chromameter. The authors deter-mined the difference between measurements taken from oxidized and nonoxidized(addition of enzyme inhibitors) samples, and expressed the results in DL*, Da* andDb*.

BROWNING INDEX

These results are expressed in Tables 9.2 and 9.6 (Mattila et al., 1993). Evaluationwas based on sensory evaluation by a trained panel.

Twenty potato slices (5 mm thick) were left to stand at 23°C for 30, 60 and 120min, and discoloration was evaluated by comparison with slices that had just beencut. Results were scored by browning grades from “0” (no color change) to “3”(strong change). To each browning grade a coefficient was attributed as follows: “0”browning grade ⇒ coefficient 0; “1” browning grade ⇒ 1; “2” browning grade ⇒5; and “3” browning grade ⇒ 10.

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As an example of a browning index calculation, consider that from the 20 potatoslices evaluated, 10 received a grade “0”, five a grade 1, three a grade 2 and two agrade 3. The final Browning Index would then be as follows:

LOSS OF REFLECTANCE

These data are presented in Table 9.5 (Ponting et al., 1972). Reflectance measurementswere made by reading total reflectance from apple slices rotated to three positions(~120° apart) and then averaging these readings to obtain the final reflectance value.Such a result was compared to readings from fresh-cut apple slices to calculate percentreflectance loss. The authors found that for apple slices, loss of reflectance correlatedbetter with the subjective evaluation of color than the “a” or “b” values.

ESTIMATE OF APPLE SUSCEPTIBILITY TO BROWNING

Amiot et al. (1992) measured the following:

1. Absorbance at 400 nm of an apple extract containing soluble pigmentformed during the browning reaction. [For details on the methods used,refer to Amiot et al. (1992).]

2. Lightness (L* ) of the pellets obtained after centrifugation during thepreparation of the soluble pigments extract. (L* results were related to theinsoluble brown pigments.)

The authors suggested that the normalized sum of A400 and L* be used to expressthe degree of browning.

Visual observation of browning is poorly correlated with measurements of absor-bance at only one wavelength (Nicolas et al., 1993). Depending on the pigmentsformed during browning, there may be a wide variation (360–500 nm) of maximumoptical absorption (Amiot et al., 1997). For a detailed discussion on measuments ofbrowning, refer to Nicolas et al. (1993) and Macheix et al. (1990).

VISUAL EVALUATION OF BROWNING

For a visual evaluation of browning potential, which can be helpful in selectingcultivars with lower browning tendency, a quick assay was described by Kader andChordas (1984).

1. PPO activity evaluation: to slices (3–4 cm diameter) of fruit or vegetable,add one drop of a freshly prepared 0.1 M solution of catechol in 0.1 Mcitric acid-phosphate buffer pH 6.2 (PPO substrate). Let rest for 6 minand then compare the samples and score the browning intensity. Thediscoloration is rated on a progressive 1 to 5 scale.

According to the authors, the natural PPO substrate content does notinterfere with the test within the 6 min duration of the assay.

10 0×( ) 5 1×( ) 3 5×( ) 2 10×( )+ + + 40=

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2. PPO substrates evaluation: to slices (3–4 cm diameter) of fruit or vege-table, add one drop of each of the following solutions in succession: a10% sodium nitrite, 20% urea and 10% acetic acid. Let rest for 4 minand then apply two drops of 8% sodium hydroxide solution.

This test is based on a color reaction developed by endogenous phenoliccompounds with the reactives added to the fruit or vegetable slice. Theintensity of the deep cherry-red color developed during the reactiondepends on the amount of phenolic compounds present in the tissue. Theresult is rated on a 1 to 5 scale, from the less colored to the most intenselycolored sample, according to the chart presented in the cited paper.

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Sapers, G.M., Garzarella, L. and Pilizota, V. 1990. “Application of browning inhibitors to cutapple and potato by vacuum and pressure infiltration.” J. Food Sci. 55:1049–1053.

Sargent, S.A., Brecht, J.K., Zoellner, J.J., Baldwin, E.A. and Campbell, C.A. 1994. “Ediblefilms reduce surface drying of peeled carrots.” Proc. Fla. Sta. Hortic. Soc. 107:245–247.

Seyderhelm, I., Boguslawski, S., Michaelis, G. and Knorr, D. 1996. “Pressure induced inac-tivation of selected food enzymes.” J. Food Sci. 61:308–310.

Shewfelt, R. 1994. “Quality characteristics of fruits and vegetables.” In R.P. Singh and F.A.R.Oliveira, eds., Minimal Processing of Foods and Process Optimization. An Interface.CRC Press, Boca Raton, FL, pp. 171–189.

Tatsumi, Y., Watada, A.E. and Wergin, W.P. 1991. “Scanning electron microscopy of carrotstick surface to determine cause of white translucent appearance.” J. Food Sci.56(5):1357–1359.

Taylor, S.L. 1993. “Why sulfite alternatives.” Food Technol. 47(10):14.Tomás-Barberán, F., Loaiza-Velarde, J., Bonfanti, A. and Saltveit, M.E. 1997. “Early wound-

and ethylene-induced changes in phenylpropanoid metabolism in harvested lettuce.”J. Amer. Soc. Hortic. Sci. 122(3):399–404.

Vámos-Vigyázó, L. 1981. “Polyphenol oxidase and peroxidase in fruits and vegetables.” CRCCrit. Rev. Food Sci. Nutr. 15:49–127.

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Watada, A.E., Ko, N.P. and Minott, D.A. 1996. “Factors affecting quality of fresh-cut horti-cultural products.” Postharv. Biol. Technol. 9:115–125.

Weemaes, C., Ludikhuyze, L., Van den Broeck, I. and Hendrickx, M. 1999. “Kinetic studyof antibrowning agents and pressure inactivation of avocado polyphenoloxidase.” J.Food Sci. 64(5):823–827.

Whitaker, J.R. 1994. Principles of Enzymology for the Food Sciences, 2nd ed., Marcel Dekker,New York.

Whitaker, J.R. 1996. “Enzymes.” In O.P. Fennema, ed., Food Chemistry, 3rd ed., MarcelDekker, New York, pp. 431–530.

Whitaker, J.R. and Lee, C.Y. 1995. “Recent advances in chemistry of enzymatic browning:an overview.” In C.Y. Lee and J.R. Whitaker, eds., Enzymatic Browning and ItsPrevention. ACS Symp. Ser. 600, Washington, D.C., pp. 2–7.

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Application of Packaging and Modified Atmosphere to Fresh-cut Fruits and Vegetables

Tareq Al-Ati and Joseph H. Hotchkiss

CONTENTS

IntroductionModified Atmosphere Packaging

IntroductionCurrent StatusShortcomings of MAP

MAP Effect on Microorganisms O

2

EffectsCO

2

EffectsMAP Effects on Respiration

PermeabilityBackground

Fick’s LawHenry’s LawTime Lag

Factors Affecting PermeabilityTemperature Effects on MAP of FCF Systems Permselectivity

IntroductionFactors Affecting PermselectivityCO

2

/O

2

Permselectivity and Respiration Quotient Role of Permselectivity in MAP

Mathematical Predictive ModelsIntroductionSteps of Developing Mathematical ModelsPublished Mathematical Models in MAP StudiesCase Study of Fresh-cut Apples

ConclusionsReferences

10

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INTRODUCTION

As market demand for fresh and minimally processed fruits increases, quality andshelf life issues increase in importance. A key requirement is to ensure that productis able to reach the consumer with minimal quality deterioration and safety risks.This becomes problematic when we recognize that fresh-cut fruit (FCF) productsconstitute a delicate, dynamic system that is prone to many forms of deterioration.In order to investigate how packaging impacts deterioration, we must first understandits causes.

The inherent quality deterioration of FCF is largely due to cutting, because itinitiates the physiological and biochemical changes at a faster rate than in intact rawfruits (Kim et al., 1993a). Generally, quality deterioration (color, flavor and texture)is attributed to the combined effect of endogenous enzymes, enhanced respiration,microbial growth (Gil et al., 1998; Kim et al., 1993b), physical abuse and environ-mental factors (Chau and Talasila, 1994). The surrounding environmental factorssuch as temperature, humidity, atmospheric composition and ethylene concentrationdirectly influence the deterioration process. Mechanical bruises and damage causedby harvesting, handling, storage and transportation are also detrimental to the FCFshelf life (Chau and Talasila, 1994).

There are no simple answers and no single treatment is known to limit overallquality deterioration. However, there are several strategies that are being imple-mented in order to reduce the rate of deterioration for FCF. These include, but arenot limited to starting with high-quality raw produce, implementing sanitationpractices, controlling temperature, lowering respiration rate, lowering ethylene pro-duction, and preventing mechanical abuse. Packaging technology is the commondenominator that allows us to implement these strategies and, thus, is key to qualitypreservation.

MODIFIED ATMOSPHERE PACKAGING

I

NTRODUCTION

A promising packaging technology for addressing quality deterioration issues ofFCF is modified atmosphere packaging (MAP). This technology is targeted at reducingthe respiration rate of fresh produce and slowing senescence. The nineteenth centuryFrench chemist Berard is reported to be the first to study the effect of modified atmo-sphere on the shelf life of horticultural products. The result of his work showed thatfruits do not ripen in anaerobic conditions. In the 1920s, Kidd and West studied howO

2

and CO

2

affect the shelf life of apples, pears and berries. Their research led tothe important finding that low O

2

and moderately high CO

2

storage conditions couldextend the shelf life of fruits.

“Modified atmosphere packaging” describes altering the gases surrounding a com-modity producing an atmospheric composition different from that of air. The purposeof modified atmosphere is not necessarily to create a fixed gas composition throughout

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Application of Packaging and Modified Atmosphere

307

the shelf life, as in controlled atmosphere storage. MAP creates a predetermined gascomposition, which may change over time. Variables that are related to produce phys-iology (respiration rate, etc.), physical factors of the environment (temperature, RH,etc.) and barrier properties of the packaging material determine the specific gascomposition at equilibrium. MAP has been commercially applied for a variety ofwhole fresh produce and minimally processed vegetables but has not achievedcomparable success for FCF for reasons that will be discussed below.

Generally, modified atmospheres can be achieved passively or actively. A passiveMAP occurs when fresh produce is hermetically sealed in a semipermeable container.The respiration process of the produce and, to a certain extent, the microbial growth,combined with container permeability alters the gas composition. Because of therespiration process, the produce consumes the surrounding O

2

and produces CO

2

,and therefore, the O

2

level is reduced, while the CO

2

level is increased. After a periodof adjustment between respiration rate and permeation rate, a steady state is estab-lished inside the package. At this stage, the amount of O

2

consumed and CO

2

produced inside the package equals the O

2

and CO

2

amount permeating through thefilm. Thus, passive atmosphere modification is a complex process with many inter-actions among different components and variables. If the container is impermeableto gases, then theoretically, O

2

concentrations may be lowered (from atmospheric21%) to near 0%, while CO

2

(initially 0.03% in the container) concentrations canreach 20% or higher.

On the other hand, an active MAP can be achieved by flushing out the air withinthe package with a precise mixture of gases to create an initial atmosphere. Usually,nitrogen serves as a filler gas to provide a precise concentration of other gases in thepackage, as well as to prevent the package from collapsing.

A fundamental knowledge of gas permeability characteristics of the film as wellas of the produce is essential for the development of both types of MAP systems.A successful MAP design requires the determination of respiration rate and respirationquotient ( ) of the produce, an appropriate polymeric film with a suitablebarrier properties, appropriate film area, appropriate produce weight, and appropriateheadspace volume.

MAP technology is an interactive system that permits interplay between thephysiological parameters of the commodity and the film characteristics. In such asystem, there are four main processes occurring simultaneously: respiration of theproduce, transpiration of the produce, permeation of gases through the packagingmaterial and heat transfer (Chau and Talasila, 1994). Respiration changes accordingto the following factors: temperature; produce maturity; and CO

2

, O

2

and ethylenelevels within the package. The temperature of the produce is also altered due tothe heat generated by the respiration process. Transpiration depends on the producesurface temperature and the temperature and relative humidity (RH) of the surround-ings. Permeability properties of the polymeric film depend on the chemical makeupof the material, ambient temperature, film thickness, permeating gas and the differ-ence in gas concentrations across the film. Understanding the extent of all of thesevariables is necessary to optimize the effect of MAP on extending the shelf life ofa chosen FCF commodity.

RCO2/RO2

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Current Status

Advances in the chemistry and engineering of polymers contribute in the develop-ment of materials for MAP applications. By far, oriented polypropylene (OPP) isthe most used material for MAP, and the majority of MAP of fresh produce ismarketed in pouches. However, MAP systems have been utilizing perforated, thin,low-density polyethylene (LDPE) for bagged produce for a long time, as well asmonolayer polyvinyl chloride (PVC) for tray overwrapped produce. Coextrusiontechnology has improved material design and properties to meet some of the MAPsystem needs. Blends of linear low- and medium-density PE with ethylene-vinylacetate (EVA) copolymer are acceptable candidates for MAP application, becausePE resins provide excellent shrink and are good moisture barriers, while the EVAcopolymer provides sealability and a higher O

2

permeability than PE resin (Robertson,1993). Nonetheless, recent research focuses on the effect of perforation on MAPapplication to fresh produce. Some studies suggest the application of perforation andmicroperforated films to prevent anaerobic conditions (Hirata et al., 1996; Lee et al.,1996; Emond et al., 1991; Hobson and Burton, 1989), while others suggest ceramic-filled plastic films (Wang et al., 1998; Lee et al., 1992). Because of the limited rangeof gas permeability of plastic films and permselectivity, which do not satisfy andmatch the wider ranges of produce respiration, all of these materials, and others,have had only limited success in MAP, especially when the produce packaged is FCF.

Shortcomings of MAP

Developments in materials science and engineering have not fully met the packagingrequirements of fresh produce in general, and FCF in particular. For example,commonly available polymeric films have a wide range of permeability coefficientsfor each individual gas that can be in the range of 1000-fold difference. However,the range of respiration rates of fresh fruit is in the range of only 10-fold. Therefore,it is possible for the films to satisfy the need for either O

2

or CO

2

alone but not bothof them at the same time. This presents one of the major disadvantages of polymericfilms: they permeate CO

2

at higher rates than O

2

. The ratio of CO

2

-to-O

2

permeabilitycoefficient is 4–6:1 which can contribute to shortening the shelf life of the productsby not maintaining the required gas atmosphere inside the package, and thus, itdefeats the purpose of the modified atmosphere created in the packages. Engineeredproperties of most recently developed polymeric materials do not address the CO

2

-to-O

2

permeability ratio, which is a crucial component for successful MAP systems,and especially so for MAP of FCF. To our knowledge, no successful solution of thisproblem has been introduced. The ultimate solution is to develop a systematicapproach that can be followed during the synthesis of polymeric materials that canproduce a wide range of permeabilities and CO

2

:O

2

permeability ratios. This wouldlead to successful materials for specific MAP-FCF products.

MAP E

FFECT

ON

M

ICROORGANISMS

Effects of gas composition created in MAP systems on microflora has been reviewedand studied intensively by many researchers (Varoquaux and Wiley, 1994; Brackett,1994). Microbial growth is a leading mechanism of deterioration and a primary

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Application of Packaging and Modified Atmosphere

309

safety factor for fresh produce products and is affected by the gas compositioncreated in a MAP system. Under inappropriate gas composition, spoilage is charac-terized by undesirable sensory changes in color, texture, flavor or odor and thepotential for growth of pathogenic microorganisms. MAP, however, can delay andarrest microbial spoilage but may not necessarily improve the produce quality.Parameters affecting microbial growth include the intrinsic properties of the food(nutrient availability, pH and a

w

) and external factors imposed by the surroundingenvironment, including the gaseous composition of the surrounding environmentand temperature (Parry, 1993).

O

2

Effects

When a sufficient O

2

level is maintained in the package to prevent anaerobic respi-ration in the produce, aerobic pathogens may grow if neither inhibitors nor compet-itors are present. The O

2

level in the package is affected by processing conditions,produce temperature, packaging permeability to O

2

, O

2

consumption rates due tothe microbial growth and produce respiration. Figure 10.1 shows how the internalO

2

pressure affects the O

2

permeability of a polymeric film and respiration rate. O

2

effects on microbial growth depend on microorganism type. In the absence of O

2

,growth of Gram-negative, aerobic spoilage organisms such as

Pseudomonas

isrestricted, while growth of Gram-positive, microaerophilic species such as

Lacto-bacillus

or

Brothothrix

flourishes. Anaerobic conditions have little effect on facul-tative anaerobes (Gram-positive or Gram-negative) (Labuza et al., 1992).

FIGURE 10.1

Oxygen consumption and permeation rates as a function of internal packageoxygen pressure. represents the critical oxygen level the package will attain. (Repro-duced with permission from Labuza, T. et al., 1992.)

P*O2

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

CO

2

Effects

CO

2

is the most important gas in MAP applications. The inhibitory effect of CO

2

on microorganisms’ growth is a complex phenomenon and is not completely under-stood. Microorganisms found under CO

2

atmosphere are different from those foundin air. As temperature increases (for example, under abuse), CO

2

protection againstmicrobial growth decreases. CO

2

in excess of 5% v/v inhibits growth of many foodspoilage bacteria, especially psychotropic species, which grow on wide range ofrefrigerated foods (Hendricks and Hotchkiss, 1997). Gram-negative bacteria aregenerally more sensitive to CO

2

than are Gram-positive bacteria. Most mold speciesrequire O

2

and are sensitive to high levels of CO

2

. Many yeasts grow anaerobicallyand are relatively resistant to CO

2

. The extent of CO

2

activity depends on the type,number and age of microorganisms, as well as CO

2

concentration, a

w

and pH of theproduct, and storage temperature. However,

C.

botulinum

and

C. perfringen

are notgreatly affected by the presence of CO

2

and are found to grow if MAP providesanaerobic conditions. Redox potential is not often considered in MAP, mainlybecause it is difficult to measure. Redox potential effects on microorganisms aremainly due to the presence or absence of O

2

and/or CO

2

(Hanlin et al., 1995).

MAP E

FFECTS

ON

R

ESPIRATION

Fresh fruits deteriorate as a consequence of respiration (Zagory, 1995). In the absenceof O

2

, anaerobic respiration occurs and generates off-flavors, off-odors and metabolictissue damage, and eventually, the tissue dies from substantial O

2

deprivation. Whenthe partial pressure of O

2

drops around 10 KPa, anaerobic respiration occurs toproduce CO

2

and ethanol. Reduced metabolites associated with offensive odors areproduced by reoxidation of reduced pyridine nucleotides, NADH and NADPH(Flodin et al., 1999). Aerobic metabolism, on the other hand, results in undesirabletextural and flavor changes due to the consumption of sugar, starch or other energystorage products of the fruit tissues. Also, one of the respiration by-products is watervapor, which upon condensation in the package, becomes free water promoting thegrowth of spoilage and pathogenic microorganisms. Another by-product of respira-tion is heat, which may reduce the effectiveness of all the measures taken fortemperature control during processing, distribution, marketing, etc. So, the higher therespiration rate, the faster the release of energy required to drive the metabolicprocesses, and therefore, the shorter the shelf life.

Successful MAP balances reduction in respiration against anaerobic metabolism.The O

2

consumption rate is balanced with the O

2

permeability of the packaging film,while maintaining the steady state O

2

at high enough levels to prevent anaerobicrespiration and at low enough levels to inhibit the respiration rate of the produce.However, in MAP of FCF, the consequences resulting from processing must also betaken into account. Because of wound response, respiration rate of apple slices, forinstance, is considerably higher than that of whole apples (Gunes et al., 2001). Whenthe respiration rates of different cultivars of apples were studied, cut apples alwaysshowed higher respiration rates than intact apples (Kim et al., 1993b). This increasedrespiration rate of FCF imposes many challenges for the MAP systems and pack-aging materials.

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Application of Packaging and Modified Atmosphere

311

PERMEABILITY

B

ACKGROUND

Nonporous polymeric films allow gases and vapors to pass through them as a functionof partial pressure differences of permeants across the films. This property of filmsis important, because it determines the atmosphere composition inside the containerwith MAP. Permeation is considered to be a solution-diffusion process. During thepermeation process, a permeant goes through three steps in the following order:condensation and mixing (solution) of the permeant in the surface of a film, migration(diffusion) to the opposite surface of the film under a concentration gradient andevaporation from the surface into the ambient surrounding (Rogers, 1985).

The permeability coefficient (P) is the product of a solubility coefficient (S), thethermodynamic parameter, and diffusion parameter (D), the kinetic parameter.Therefore,

P

=

DS (1)

The solubility coefficient indicates how much gas a polymeric film can take up,and it is measured by the concentration (c) of the sorbed gas per unit volume of thefilm. The polymer-permeant interactions, inherent condensibility of the permeantand the amount of free volume in a glassy polymer determine the solubility coeffi-cient (Kim et al., 1988). The diffusion coefficient (D) accounts for the mobility ofthe permeant molecules in the film. In the macroscopic picture, the sorbed gasmolecules move under the force of the chemical potential gradient of the permeatinggas in the membrane (

µ

/

x). At position x within the film matrix (0

x

l

, where

l

=

thickness of the membrane), the flux density of the permeant (

J

) is expressedby Fick’s law according to the following:

J

x

=

D (

C/

x) where D

=

constant.

Fick’s Law

According to Fick’s law, gas diffuses, under steady state, through polymeric materialsat a constant rate as long as a constant pressure difference is applied across thematerial. The amount of the permeant moving through the film of unit area in a unittime, is diffusive flux or transport rate J:

J

=

Q

/

A t (2)

where (Q) is the total permeant amount passing through area (A) during time (t).However, the concentration gradient is proportionally related to the (J) in Fick’s lawas follows:

(3)

where (D) is diffusion coefficient, c is the concentration of the permeant and isthe concentration gradient of the permeant across a given thickness (

x).

J D∂c∂x------–=

∂c∂x------

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Given that (D) is a constant and is independent from (c), then once the steadystate is established, (J) becomes a constant, and Equation (3) can be integratedacross the total thickness of the polymer film (x) and between the two concentrations(c

1

and c

2

), so:

(4)

Substituting for J [Equation (2)] into Equation (4), the permeant amount diffusingthrough a film of area (A) and time t is as follows:

(5)

Henry’s Law

Because the permeant is a gas, it is more convenient to replace concentration withpartial pressure (p) of the permeant gas. So, at low concentrations, (c) can beexpressed, according to Henry’s law, as follows:

c

=

S p (6)

where (S) is the solubility coefficient of the permeant in the polymer film. Equations(5) and (6) can be combined as follows:

(7)

where p

1

is the pressure of permeant in the high-pressure side of the film, and p2 isthe pressure in the lower-pressure side of the film.

Permeability coefficient (P) is shown in Equation (7) as the product (DS), so:

(8)

(9)

Time Lag

Permeability measurement can be carried out experimentally using the time lagmethod. From the typical permeation curve (Figure 10.2), during the steady state, theamount of gas permeating through the polymeric film increases linearly with time.The linear portion of the steady state line AB can be extrapolated back to Q = 0,

Jx D c2 c1–( ) and J– Dc1 c2–( )

x--------------------= =

Q Dc1 c2–( ) A t

x-----------------------------=

Q D Sp1 p2–( )A t

x----------------------------=

PQx

A t p1 p2–( )----------------------------=

Qt---- P

x--- A ∆p( )=

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Application of Packaging and Modified Atmosphere 313

where the intercept t = L. However, when the film initially is penetrant-free, thefollowing has been shown:

(10)

The extrapolated steady state line intercepts with the time axis at a point repre-sented in the graph by value L, referred to as time lag. All three parameters of thepermeation process (P, D, S) can be calculated from one permeation measurementtest. The slope of the line (A-B) represents the permeation rate at steady state, i.e.,(Q/t). If Q/t is substituted in Equation (9), (P) can be calculated. From the time lag L,(D) can be calculated according to Equation (10), and (S) can be calculated from(S = P/D). It has been found that the steady state is almost always reached after aperiod of time that is equivalent to approximately 2.7 L (Rogers, 1985).

FACTORS AFFECTING PERMEABILITY

Film barrier properties combined with respiration determine gas composition insideMAP systems of FCF and are, in turn, affected by the basic structural and chemicalproperties of polymeric films and, thus, play important roles in controlling the perme-ation process. Understanding the influence of polymer structure aids in understanding

FIGURE 10.2 Typical permeation and time lag curve from a permeability experiment.Amount of permeated penetrant, Q, as a function of time, t. Extrapolation of the steady stateline AB to the time axis gives the lag time as intercept L. (With permission from Rogers,C.E. 1985.)

Dx2

6L------- and L

x2

6D-------= =

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314 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

the fundamental causes of the limitations of current polymer films (see “Shortcomingsof MAP” presented earlier). More research can then be directed toward engineeringbetter-suited polymeric films to meet the challenges imposed by MAP of FCF.

Several factors affect permeability of polymeric films (Ashley, 1985). Suchchemical groups as nitrile, fluoride, chloride, acrylic and ester induce polarity inchain segments, which increases chain packing and, thus, reduces permeability. Forexample, polar polymers that contain hydroxyl groups as polyvinyl alcohol havevery low gas permeability, whereas those with nonpolar groups as polyethylene havehigher gas permeability. Linear, simple molecules promote chain packing, and thus,the polymer would acquire lower permeability, whereas polymers in which thebackbones contain bulky side groups have weaker chain packing ability and, thus,higher permeability. Crystallinity also plays an important role in affecting the per-meability, as crystallites are impervious to gases, making the amorphous areas ofpolymers the only regions for the permeation process to occur. The higher thecrystallinity, the lower the permeability. In addition, orientation can play an importantrole for amorphous polymers. Orientation of polymer chains in the amorphous regionmay lead to about 10–15% reduction in permeability. Cross-linking between polymerchains inhibits permeant transport, as cross-links reduce the diffusion coefficients(Chodak, 1995).

Polymeric materials can exhibit either rubbery or glassy state depending on thetemperatures at which the materials are used. The transition from one state to anotheroccurs at a glass transition temperature, Tg (Vieth, 1991; Ganesh et al., 1992;Mandelkern, 1972). At a high enough temperature, a polymer exhibits a rubber,liquid-like state with oscillated molecular chains occupying the amorphous region.Rubbery polymers are tough and flexible due to the free mobility of the polymerchains with random conformations. As the temperature is lowered, polymer mole-cules exhibit a well-organized crystalline structure. At low enough temperatures,there is no adequate mobility for the polymer chains to achieve their equilibriumconfigurations; hence, the polymer exhibits a glass phase. Glassy polymers are brittleand hard due to the restricted segmental mobility. Tg temperatures for commonpolymers vary widely, and for many, the Tg is well above room temperature.

The permeability of rubbery and glassy polymers is a function of the molecularmass of the permeant (Figure 10.3) (Bell et al., 1988). Rubbery polymers behavedifferently from glassy polymers in terms of their permeability to different gases.There are two important phenomena observed in this figure. The permeability coef-ficients of rubbery polymers increase as the permeant molecular mass increases, andmeanwhile, the permeability coefficient of glassy polymers decreases as the permeantmolecular mass increases. This phenomenon can be explained as follows. With increas-ing molecular mass of gases, while gas solubility coefficients increase in both poly-mers, the diffusion coefficient of gases decreases more rapidly in glassy polymersthan in rubbery polymers. However, the increase in solubility coefficient has moreeffect than the decrease in diffusion coefficient for rubbery polymers, but it has lessprofound effects than the decrease in diffusion coefficient for glassy polymers. There-fore, gases with smaller molecular mass would have higher diffusion coefficients and,therefore, would permeate preferentially in glassy polymers. While in glassy poly-mer, gas molecules with higher solubility coefficients would permeate preferentially.

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Application of Packaging and Modified Atmosphere 315

TEMPERATURE EFFECTS ON MAP OF FCF SYSTEMS

The effect of temperature on MAP of FCF is twofold. It affects the respiration rateof FCF and the permeability of the packaging materials. Due to the unpredictabilityof temperature changes and difficulties in maintaining an optimum temperature,temperature often plays a detrimental role on shelf life of FCF under MAP condi-tions. Both permeability and respiration rate follow an Arrhenius-type relationshipas a function of temperature (Mannapperuma and Singh, 1994) as follows:

P = Po exp (−Ep /R*T)

R = Po exp (−ER /R*T)

FIGURE 10.3 Permeability coefficient, P, of gases in —, �, �: LDPE, and ---, �, �:polycarbonate. (Reprinted from Journal of Membrane Science, Bell et al., “Selection ofPolymers for Pervaporation Membranes,” 36: 315–329, Copyright 1988, with permission fromElsevier Science.)

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316 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

where P = permeability, Po = Arrhenius constant, R = respiration rate, Ro = Arrheniusconstant, EP = activation energy of permeation, ER = activation energy of respiration,R* = universal gas constant (J/mol-K) and T = temperature (K).

According to the Arrhenius equation, both respiration and permeability have aproportional relationship with temperature. The equations further suggest that tem-perature would have a negligible effect on gas composition within MAP of freshproduce if both permeation and respiration rates are increased by the same magni-tude. However, this relationship may not hold true for MAP of FCF due to the factthat cut fruits usually have higher respiration rates and therefore, the response totemperature is expected to be more profound than the effect on permeability. There-fore, as the temperature increases, the O2 level inside the package is expected todecrease to a greater extent than the Arrhenius equation predicts. In this case, therespiration rate of FCF consumes more O2 than the film can permeate into thepackage, even with increased permeability coefficient. This raises concerns associ-ated with anaerobic development and importance of temperature control for MAPof FCF. Other elements of deterioration such as ethylene production, transpirationand microbial growth are also affected by temperature (Chau and Talasila, 1994).Cooling fresh produce immediately after harvest and holding at an appropriate lowtemperature throughout the processing of the FCF, transportation, marketing andpostpurchasing are among the most important steps to minimize losses and preservequality. Equally important, the barrier properties should function to counteract theeffect of the increased respiration rate of FCF for a given MAP system.

PERMSELECTIVITY

INTRODUCTION

Film CO2/O2 permselectivity (β) is the ratio of the CO2 permeability coefficient to O2 permeability coefficient

(11)

As pointed out above, the dynamics of permeability and permselectivity character-istics of a gas mixture/polymer system are eventually determined by the diffusionand solution properties of a polymeric film. Differences in permeability coefficientsamong different films are generally attributed to differences in their diffusion coef-ficients more than solubility coefficients. However, permselectivity of films to gasesis mainly dependent on diffusion coefficients for glassy polymers and on solubilitycoefficients in the case of rubbery films. Figure 10.4 shows the typical behavior ofpolymers in which permeability is inversely related to permselectivity. Rubbery andglassy polymers with low CO2 and O2 permeability coefficients have relatively higherCO2/O2 permselectivity than those polymers with higher permeability coefficientsfor this gas pair (Petropoulos, 1990).

(PCO2) (PO2

):

β PCO2/PO2

=

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Application of Packaging and Modified Atmosphere 317

FACTORS AFFECTING PERMSELECTIVITY

For a pair of gases, permselectivity is based on the diffusivity selectivity and solubility selectivity of a polymeric film, and according to Equations(1) and (11):

(12)

The diffusivity selectivity relies on the polymer segmental mobility and interseg-mental packing of the polymer chains and the differences in size and shape of thetwo penetrants. The film functions as a selective media for the size and shape ofthe penetrant pair, whereas the solubility selectivity relies on the differences of thecondensibility between the gas pair and the physical interactions between the gases

FIGURE 10.4 Permselectivity (CO2/CH4)-permeability (CO2) relation for some commonglassy (�) and rubbery (�) polymers (KA = Kapton polyimide; UL = Ultem polyetherimide;PMA = polymethylacrylate; CA = cellulose acetate; EC = ethyl cellulose; NR = natural rubber;SR = silicone rubber; MR = methyl rubber; PPSX = polyphenyl siloxane; PC = polycarbonate;PSF = polysulfone; PPO = polyphenylene oxide; PMMA = polymethyl methacrylate. Filledpoints: PMDA-ODA (1), PMDA-IPDA (2) and 6FDA-ODA (3). (Reprinted from Journal ofMembrane Science, Petropoulos, J.H. “Some Fundamental Approaches to Membrane GasPermeability and Permselectivity,” 53: 229–258, Copyright 1990, with permission fromElsevier Science.)

(DCO2/DO2

)(SCO2

/SO2)

βPCO2

PO2

----------DCO2

DO2

-----------SCO2

SO2

----------= =

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318 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

and the polymer chains (Kim et al., 1988). It is important to understand the differ-ences between glassy and rubbery polymeric films in order to explore the permse-lectivity phenomenon. Glassy polymers have a low intrasegmental mobility and longrelaxation times, which is not true for rubbery polymers. Rubbery polymers havelow solubility coefficients for gases with low critical temperatures, so the diffusion,solubility and permeability coefficients are independent of the gas pressure or con-centrations (assuming no plasticization). This explains, for instance, the much highersolubility coefficient for CO2 in LDPE relative to O2 that even higher O2 diffusioncoefficients cannot overcome; therefore, CO2 always has a higher permeabilitycoefficient than O2 in most rubbery and glassy polymers. However solubility, diffu-sion and permeability coefficients are functions of gas pressures for glassy polymers.The structure/permeability relationship can explain the differences between theglassy and rubbery polymers. Most of the differences in behaviors are attributed tothe fact that glassy polymers are not usually in the true thermodynamic equilibriumstate, and so the structure effects on permeability of rubbery and glassy polymersare different (Stern, 1994).

Examples of studies aimed at altering CO2/O2 permselectivity for MAP purposesare rare. The difficulty arises from the inherent high solubility of CO2 in most ofthe plastic films relative to O2, despite the higher O2 diffusion coefficient. However,Kim et al. (1987) have attempted successfully to reverse permselectivity of N2 andCH4. Except for polysulfone, with N2/CH4 permselectivity of 1.0, most plastic filmshave permselectivity values less than 1.0 for this pair. For instance, the N2/CH4 is0.27 for natural rubber and 0.93 for polycarbonate. Because CH4 has a higher criticaltemperature (191°K) than N2 (126°K), it is more condensible than N2; therefore,CH4 has a higher solubility coefficient than N2. So, the CH4 permeability is higherthan the N2 permeability, despite the fact that the diffusion coefficient of N2 is slightlyhigher than CH4 in both rubbery and glassy polymers. However, manipulation ofthe chemical structure of the polyimide family leads to an increase in the N2 perme-ability so that polyimides can have a range of N2/CH4 permselectivity all of whichis higher than 1. For example, PMDA-ODA has an N2/CH4 permselectivity of 1.75,6FDA-IPDA, 1.9, and 6FDA-ODA, 2.18, etc. (Kim et al., 1987).

Additionally, although not investigated for MAP applications, there are severalfactors believed to affect the permselectivity (Stern, 1994). For instance, chain stiff-ness of the backbone chains can be increased by introducing bulky groups that inhibitintrasegmental mobility (rotation), thus reducing the permeability and increasing thepermselectivity. Another approach involves reducing chain packing by introducingcharges, which increases permeability. Optimizing gas atmosphere evolution insideMAP applications of different FCF products must rely on investigating such approachesand properties of polymeric films, which so far has not been comprehensively carried out.

CO2/O2 PERMSELECTIVITY AND RESPIRATION QUOTIENT

The respiration quotient (RQ) is the ratio of CO2 production rate to O2 consumptionrate and can be stated as follows:

(13)RQ RCO2/RO2

=

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Application of Packaging and Modified Atmosphere 319

Where is the CO2 production rate, and is the O2 consumption rate. If foreach molecule of CO2 produced during aerobic respiration, one molecule of O2 isconsumed, the respiration quotient (RQ) = 1. CO2/O2 permselectivity is an importantproperty of polymeric films, especially those for MAP application of FCF. Therefore,to match a film permselectivity with a given produce respiration quotient (RQ) is achallenge that would contribute to a successful application of MAP for FCF products.

If a polymeric film permeates O2 and CO2 equally (i.e., : ratio is 1:1),then when the O2 level is reduced due to produce respiration from, say, 21–3%(18% change), the package would also create an equal amount of change for CO2

as well, i.e., CO2 would increase to 18% within the package. Because differentproduce items require different O2 and CO2 levels, both high and low levels of thegases can be beneficial or harmful, depending on the variety and type of produce.In this case, permselectivity of CO2/O2 is as important as the absolute permeabilityvalues for MAP of FCF (Zagory, 1995).

ROLE OF PERMSELECTIVITY IN MAP

Despite the critical importance of permselectivity for the successful application ofMAP to whole and cut produce, little progress has been made in developing materialswith optimum permselectivities. Use of a polymeric film with a high β (e.g., >4–6)results in an equilibrium atmosphere that is low in CO2, and films with low β (e.g., <2)tend to accumulate high levels of CO2 without regard to absolute permeation rates.The effect of β on the package atmosphere cannot be overridden by any other means,including gas flushing, vacuum packaging, changing the size of the bag or changingthe amount of the product in the bag. Thus, selection of optimal β will be necessaryto optimize any packaging system for FCF.

β values can be used to predict the equilibrium concentration of CO2 and O2

inside a package, which in turn, can be compared to the optimal mixture, once therespiration rate of the produce is known. An illustration of the significance of β onMAP systems is given in Figure 10.5. The optimal ranges for O2 and CO2 concen-tration needed in a modified atmosphere for different produce commodities areplotted based on literature values (Zagory, 1995). For example, the optimal CO2

range for grapefruit is 5–9% and the O2 is 4–10%. Atmospheres within these valueswill prolong product quality. PVC, which has a β value of 6, would result in CO2

and O2 mixtures represented by line E in Figure 10.5. Thus, the maximum amountof CO2 achievable would be approximately 3%. This is well below the optimum forgrapefruit. It would be possible, however, to achieve an optimum gas atmospherefor oranges (Figure 10.5).

The range of values for β in common films is more limited than the range ofrecommended O2 and CO2 concentrations, meaning that most produce items do nothave a matching suitable film to produce an optimal atmosphere. Currently availablefilms satisfy only whole fresh produce with relatively low O2 and CO2 requirements.Thus, commonly available polymeric films fail to provide the recommended atmos-phere for most fresh whole produce items and are woefully inadequate for FCFproducts due to their higher respiration rates. For instance, a film with β value of3.4 (e.g., PET) passes through recommended areas O2 and CO2 for orange and

RCO2RO2

PCO2PO2

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320 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

avocado (Figure 10.5) and would be appropriate for those two commodities. Becausethe β line of PET does not pass through cherries, the film is not the appropriate filmwith which to package this commodity.

Perusal of the permselectivities for a large number of polymeric films (Table 10.1)show a relatively limited of selectivity of 2–10, with most common films in therange of 4–6. This means that the produce in a MAP system must have a respirationrate that would match β values of 4–6 to produce an optimum atmosphere. The rangeof available selectivities is too limited for the much wider range of respiration ratesamong produce.

MATHEMATICAL PREDICTIVE MODELS

INTRODUCTION

To design an optimal MAP system for a given FCF (or whole fruits or vegetablesfor that matter), it is necessary to predict changes in atmosphere composition overtime inside the package. Such prediction is possible with the help of mathematicalmodels. Such models facilitate selecting an appropriate packaging material for agiven FCF, once packaging dimensions and respiration rate are known, by calculatingthe required permeability coefficients of the packaging material and then choosingmaterials with comparable gas permeability. If the permeability and respirationcharacteristics are provided, the dimension of the package can be calculated as well.

Several models have been published (Senesi et al., 1999; Cameron et al., 1995;Piergiovanni et al., 1999; Lee and Renault, 1998; Lakakul et al., 1999; McLaughinand O’Beirne, 1999; Ishitani and Inoue, 1993; Chinnan 1989; and Mannapperumaand Singh, 1994). For any MAP system, what determines O2 influxes and CO2

FIGURE 10.5 Recommended gas composition for fruits and permselectivity of various poly-mer films. (Reproduced with permission from Zagory, D. 1995.)

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Application of Packaging and Modified Atmosphere 321

TABLE 10.1CO2 and O2 Permeability Coefficients and CO2/O2 Permselectivity of Various Polymeric Materials

Material Permeability Coefficient Permselectivity

No. Type CO2 O2 Units CO2/O2 Ref.

1 LDPE 99 27 3.7 Cameronet al., 19952 PP 58 9 6.2

3 PVC 0.65 0.19 3.44 Cellulose acetate 348 10 34

5 PSS-Na1 14.1 2.9 Barrer* 4.9 Chen and Martin, 1994

6 PSS-Mg2 8.9 1.8 4.9

7 Poly(ethyl methacrylate)

7 × 10−10 1.9 × 10−10 3.7 Chiou and Paul, 1989

8 TMPC/SAN3 6.56 × 10−6 7.9 × 10−7 8.3 Chiou and Paul, 1987a

9 Cellulose acetate 9 0.92 Barrer Feldmanet al., 1987

10 PSF4 5.5 1.29 Barrer 4.3 Ghosal et al., 199511 PSF-NO2 (50%) 3.4 0.88 3.9

12 PSF-NO2 (98%) 2.3 0.66 3.513 PSF-NO2 (192%) 1.5 0.44 3.4

14 PC5 6.8 1.6 Barrer 4.3 Hellmuset al., 198915 TMPC 18.6 5.6 3.3

16 HFPC6 24 6.9 3.417 TMHF-PC7 111 32 3.5

18 Polyimide 1357 213 Barrer 6.4 Hofman,et al., 1996

19 PSF 5.6 1.4 Barrer 4 Houde et al., 199420 PSF-PPHA8 6.5 2 3.3

21 PMMA 3.1 × 10−11 8.75 × 10−12 3.5 Chiou and Paul, 1987b22 CO2-PMMA9 4.91 × 10−11 1.26 × 10−11 3.9

23 Vulcanized rubber

0.132 × 10−6 0.02 × 10−6 6.6 Barrer, 1939

24 PE @ 15oC 130 27.5 4.7 Soboleyet al., 195525 I08r PE10 72.7 15.3 4.75

26 PET 53 6.1 8.7 Pye et al., 1976

27 PE (1.2 mil thick)

12.9 3.4 Barrer 3.8 Pasternaket al., 1970

28 Cellophane 0.0008 × 10−6 0.0002 × 10−6 4 Davis, 194629 Koroseal11 0.26 × 10−6 0.055 × 10−6 4.730 Vinylite12 0.14 × 10−6 0.036 × 10−6 3.931 Pliofilm13 0.56 × 10−6 0.11 × 10−6 5.1

(continued)

10 13– mol m

m2 s kPa---------------------------

cm3(STP) cm

cm2 s cmHg---------------------------------

cm3(STP) cm

cm2 s cmHg---------------------------------

cm3 (STP) cm

cm2 s cmHg----------------------------------

cc mm

cm2s cmHg----------------------------

1010cc STP( ) mm

cm2s cmHg-------------------------------------------

10 12– cc STP( ) mm

cm2s cmHg---------------------------------------------

c cm

cm2 min cmHg------------------------------------

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322 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

TABLE 10.1CO2 and O2 Permeability Coefficients and CO2/O2 Permselectivity of Various Polymeric Materials (Continued)

Material Permeability Coefficient Permselectivity

No. Type CO2 O2 Units CO2/O2 Ref.

32 Mylar 346 74 4.7 Karel et al., 196333 PP 11,200 4110 2.7

34 PMSP14 280 77.3 3.6 Ichirakuet al., 1987

35 21% S-PE15 14.9 3.45 4.3 Myers et al., 196036 41% S-PE16 20.2 4.26 4.7

37 1.8% A-PE17 25.4 5.73 4.438 31% A-PE18 9.7 2.24 4.339 34% V-PE19 11.3 2.33 4.840 60% V-PE20 2.47 0.57 4.3

41 Epoxy-diacrylate-co21

1.53 × 10−3 1.19 × 10−3 1.3 Bellobono et al., 1991

42 Al3-Sil-A/U22

(@200°C)5.5 4 1.4 De Lang

et al., 199543 PMP23 10,700 2700 Barrer 4 Morisato and

Pinnau, 1996

44 PTBA24 560 130 4.3

45 PC25 6 1.5 Barrer 4 Muruga-nandam and Paul, 1987

46 TCPC26 6.7 2.3 2.947 TMPC27 4.2 1.4 3

48 Teflon AF-240028

3900 1600 Barrer 2.4 Pinnau and Toy, 1996

49 37% DMS29 2.2 1.04 2.1 Suzukiet al., 199550 57.4% DMS30 61.7 11.3 5.5

51 65% DMS31 298 49.4 6

52 t-Bu acetylene32 13.6 × 10−8 3 × 10−8 4.5 Takada and Matsuya, 1985

53 Cl-Ph acetylene33 61 × 10−10 8 × 10−10 7.6

54 6FDA-3,3′-ODA34

2.1 0.68 Barrer 3.1 Mi et al., 1993

55 6FDA-4,4′-ODA35

22 5.05 4.4

56 pp-PFP/PDMS36 20 6 Barrer 3.3 Oh and Zurawsky, 1996

57 pp-PFT/PDMS37 21.7 4.9 4.4

cc mil

m2 day atm-----------------------------

108cm3 STP( ) cm

cm2s cmHg------------------------------------------

109ccmm

cm2s cmHg----------------------------

cm3 STP( ) cm

cm2s cmHg---------------------------------

10 7– mol

m 2– s Pa---------------------

109cm3

STP( ) cm

cm2s cmHg------------------------------------------

cm3 STP( ) cm

cm2s cmHg---------------------------------

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Application of Packaging and Modified Atmosphere 323

TABLE 10.1CO2 and O2 Permeability Coefficients and CO2/O2 Permselectivity of Various Polymeric Materials (Continued)

Material Permeability Coefficient Permselectivity

No. Type CO2 O2 Units CO2/O2 Ref.

58 Uncoated PE38 9.3 3.3 Barrer 2.8 Surya-narayanan et al., 1974

59 PP coated PE39 1.9 1 2

60 Polyethylene methacrylate

4.3 Talasila and Cameron, 199761 Polyvinyl-

benzoate5.8

62 PVC (unplasticized)

3.5

63 Polytetrafluoroeth-ylene

2.3

64 Polybutadiene 7.365 Ethyl cellulose 7.766 Cellulose cetate

(plasticized)18.7

67 Polystyrene 10,000–26,000

2600–7700 3.4–3.8 Kader et al., 1989

68 Polyvinylidene 59 15.5 3.869 Polycarbonate 23,250–

26,35013,950– 14,725

1.7–1.8

70 Methylcellulose 6200 1240 5

71 Ionomer40 7.73 × 10−8 1.85 × 10−8 4.2 Del Nobile et al., 199572 Ionomer41 5.64 × 10−8 1.44 × 10−8 3.9

73 6FDA-DAF42 19.5 5.1 Barrer 3.8 Kim and Koros, 198974 6FDA-IPDA43 24.3 5.1 4.8

75 6F-TADPO44 27.6 7.9 Barrer 3.5 Koros and Fleming, 1993

76 6F-ODA45 23 4.3 5.3

77 LLDPE46 @25°C N/A N/A 4.4 Villaluenga et al., 199878 LLDPE47 @80°C N/A N/A 3.0

79 PPO48 58 18 Barrer 3.2 Koros et al., 1987

80 Ionomer49 1.21 × 10−7 3.15 × 10−8 3.8 Mensitieri et al., 1996

81 Ionomer50 1.03 × 10−7 2.61 × 10−8 3.9 Mensitieri et al., 1996

82 Ionomer51 1.13 × 10−7 3.01 × 10−8 3.8

83 PS 88 11 8 Robertson, 199384 Nylon 6 1.6 0.38 4.2

(continued)

mL mil

m2 day atm---------------------------

cm3 STP( ) cm

cm2s cmHg---------------------------------

cm3 STP( ) cm

cm2s atm---------------------------------

cm3 STP( ) cm

cm2s atm---------------------------------

1011cm3

STP( ) cm

cm2s cmHg--------------------------------------------

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324 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

TABLE 10.1CO2 and O2 Permeability Coefficients and CO2/O2 Permselectivity of Various Polymeric Materials (Continued)

Material Permeability Coefficient Permselectivity

No. Type CO2 O2 Units CO2/O2 Ref.85 PMDA-ODA52 2.7 0.61 Barrer 4.4 Kim et al.,

198886 PMDA-MDA53 4.3 0.98 4.487 PMDA-IPDA54 26.8 7.1 3.8

88 PVC/MSN55 9 Miltz et al., 199289 PVC/PA56 2.4

90 Poly(butadiene-styrene)

6.5 × 10−1 7 × 10−2 9.2 Examaet al., 1993a

91 Butyl rubber 1.6 × 10−2 3.6 × 10−3 4.492 Saran 4.63 × 10−5 4.54 × 10−6 10.2

93 Silicone rubber 7.13 1.1 6.5 Examaet al., 1993b

94 Ethyl cellulose 0.378 0.155 2.495 Natural rubber 0.672 0.095 7.196 HDPE 7.4 × 10−4 1.53 × 10−4 4.897 Rubber

hydrochloride3.67 × 10−3 1.5 × 10−3 2.4

98 Poly(vinylchloride-vinyl acetate)

1.74 × 10−1 2.62 × 10−2 6.6

* Barrer = 10−10 cm3 (STP) cm/cm2 s cmHg1 PSS-Na: Na counterion of poly(styrene-co-styrenesulfonic acid)2 PSS-Mg = Mg counterion of poly(styrene-co-styrenesulfonic acid)3 TMPC/SAN = tetramethyl bisphenol-A polycarbonate/styrene-acrylonitrile copolymer4 PSF = polysulfone; nitrate with different levels (50%, 98% and 192%)5 PC = polycarbonate6 HFPC = hexafluorobisphenol-A polycarbonate7 TMHF-PC = tetramethylhexafluorobisphenol-A polycarbonate8 PSF-PPHA = phenophthalein-based polysulfone9 Conditioned extruded poly(methyl methacrylate) under 25 atm CO210 108r PE = irradiated PE with a dose of 108 roentgens11 Koroseal = polyvinyl chloride composition12 Vinylite = polyvinyl chloride13 Pliofilm = rubber-wax composition14 PMSP = poly(1-trimethylsilyl-1-propylene)15 21% S-PE = 20.9% grafted styrene-polyethylene copolymer16 41% S-PE = 41.3% grafted styrene-polyethylene copolymer17 1.8% A-PE = 1.8% grafted acrylonitrile-polyethylene copolymer18 31% A-PE = 31.3% grafted acrylonitrile-polyethylene copolymer19 34% V-PE = 34% grafted vinylpyridine-polyethylene copolymer20 60%V-PE = 60% grafted vinylpyridine-polyethylene copolymer

mL mil

cm2•h•atm

---------------------------

mL mil

cm2•h•atm

---------------------------

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Application of Packaging and Modified Atmosphere 325

TABLE 10.1CO2 and O2 Permeability Coefficients and CO2/O2 Permselectivity of Various Polymeric Materials (Continued)

21 Epoxy-diacrylate-co = photochemically grafting an epoxy-diacrylate copolymer containing 30 wt.% ofactive carbon onto cellulose22 Al3-Sil-A/U = silica-modified γ-alumina membrane23 PMP = poly(4-methyl-2-pentyne)24 PTBA = poly(tert-butylacetylene)25 PC = bisphenol-A polycarbonate26 TCPC = tetrachloro-bisphenol-A polycarbonate27 TMPC = tetramethylbisphenol-A polycarbonate28 Teflon AF-2400 = an amorphous PDD-PTFE copolymer containing 87 mol% 2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole and 13 mol% tetrafluoroethylene29 37% DMS = poly(styrene-co-maleic anhydride) with 37% dimethylsiloxane (DMS MW = 10,000)30 57.4% DMS = poly(styrene-co-maleic anhydride) with 57.4% dimethylsiloxane (DMS MW = 10,000)31 65% DMS = poly(styrene-co-maleic anhydride) with 65% dimethylsiloxane (DMS MW = 10,000)32 t-Bu acetylene = tert-butylacetylene33 Cl-Ph acetylene = 1-chloro-2-phenylacetylene34 6FDA-3,3′-ODA = polyimide isomer (5,5′-[2,2,2-trifluoro-1(trifluoromethyl)-ethylidene]-bis-1,3-isobenzofurandione)-diamine35 6FDA-4,4′-ODA = polyimide isomer (5,5′-[2,2,2-trifluoro-1(trifluoromethyl)-ethylidene]-bis-1,3-isobenzofurandione)-diamine36 pp-PFP/PDMS = plasma polymerized pentafluoropyridine poly(dimethylsiloxane)37 pp-PFT/PDMS = plasma polymerized pentafluorotoluene poly(dimethylsiloxane)38 Uncoated PE = uncoated 1-mil PE film39 PP coated PE = 1-mil PE film coated with 1–2 microns PP40 Ionomer1 = E-0.046AA-0.12Zn [ethylene-acrylic acid (0.046%) with 0.12% Zn] @25°C41 Ionomer2 = E-0.065AA-0.25Zn [ethylene-acrylic acid (0.065%) with 0.25% Zn] @25°C42 6FDA-DAF = 5,5′-[2,2,2-trifluoro-1 (trifluoromethyl)-ethylidene]-bis-1,3-isobenzofurandione)-diamine,2,7-fluorenediamine43 6FDA-IPDA = 5,5′-[2,2,2-trifluoro-1 (trifluoromethyl)-ethylidene]-bis-1,3-isobenzofurandione) -isopropylidenedianiline44 6F-TADPO = ether connected polypyrrolone polyimide45 6F-ODA = ether connected polyimide46 LLDPE46= linear low-density polyethylene extruded with 15 mm distance between die and chill role2 cmHg vacuum, at 25°C47 LLDPE47 = linear low-density polyethylene extruded with: 5 mm distance between die and chill role,5 cmHg vacuum, at 80°C 48 PPO = polyphenylene oxide49 Ionomer49 = E-0.046AA-0.12Zn [ethylene-acrylic acid (0.046%) with 0.12% Zn]50 Ionomer50 = E-0.065AA-0.25Zn [ethylene-acrylic acid (0.065%) with 0.25% Zn]51 Ionomer51 = E-0.037AA-0.35Zn [ethylene-acrylic acid (0.037%) with 0.35% Zn]52 PMDA-ODA = polypyromellitim of oxydianiline53 PMDA-MDA = polypyromellitim of methylenedianaline54 PMDA-IPDA = polypyromellitim of isopropylidenedianaline55 PVC-MSN 55 = the difference between 55 and 56 is only plasticizer content56 PVC/PA56 = the difference between 55 and 56 is only plasticizer content

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326 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

effluxes from a given polymeric package, at a given temperature, are the permeabilitycoefficient, thickness of the film, surface area and partial pressure gradient of O2

and CO2 inside and outside of the film. There is control over the first three parameters,but the optimum partial pressure gradients are determined by the produce and aredictated by the optimum atmosphere composition for a given produce item. There-fore, an appropriate mathematical model must be carefully designed and tested takinginto account these parameters. The models are similar and are generally developedwith the same basic steps as follows.

STEPS OF DEVELOPING MATHEMATICAL MODELS

The mathematical models combine mathematical descriptions of gas fluxes throughboth polymeric film and the fresh produce (Robertson, 1993). For the film, Fick’slaw is applied. From Equation (9), CO2 and O2 flux across the film are as follows:

(14)

(15)

where Q is the diffusive flux of O2 and CO2 through a film in a unit time, isthe partial pressure of O2 outside the package, is the O2 partial pressure insidethe package, is the CO2 partial pressure outside the package and is theCO2 partial pressure inside the package.

The O2 flux into the fruit is a function of respiration rate, expressed as either or

(16)

where Qf is the flux of O2 into the fruit per unit time, is the respiration rate (oroxygen consumption) of the fruit (mL/Kg.h) and W is the weight of fruit in thepackage. Gas exchange in a MAP of a fresh produce system reaches equilibriumafter some time, when the amount of gas consumed (or produced) by the produceequals the amount of gas flux (influx and efflux) through the package. Since at thissteady state, the O2 flux through the film and into the fruit are equal, then

(17)

Once the relationship between the respiration rate ( ), weight of produce,package dimensions and O2 concentration are known, Equation (17) provides amathematical means to predict the O2 permeation requirement for a film to successfully

QO2

PO2

x------- A pO2O

pO2i–( );=

QCO2

PCO2

x---------- A pCO2i

pCO2O–( );=

pO2O

pO2i

pCO2OpCO2i

(RO2) (RCO2

):

Qf RO2W=

RO2

QO2Qf=

PO2

x------- A pO2O

pO2i–( ) RO2

W=

RO2

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Application of Packaging and Modified Atmosphere 327

perform in this MAP system. Similarly, a predictive mathematical equation to modelCO2 evolution inside the package can be developed as follows:

(18)

PUBLISHED MATHEMATICAL MODELS IN MAP STUDIES

The published models for respiring produce packaged under modified atmosphereconditions reveal an interesting fact—all models are very similar.

Jurin and Karel (1963) published one of the earliest models, which addressedthe respiration rates of ‘McIntosh’ apples as a function of oxygen concentration.They used the relationships obtained in respiration studies to predict optimum pack-aging conditions for apples. The packaging parameters considered were volume,surface area and permeability to O2 and CO2. At steady state, concentrations of O2

and of CO2 [assuming the respiration quotient (RQ) = 1] were obtained by thefollowing equations:

(19)

(20)

WhereVc = volume of O2 consumed (cc/package x day) c2 = O2 concentration in the package expressed as a fraction

Vd = O2 diffusion into the package (cc/package x day) = O2 permeability (cc.mil)/(m2.day.atm)

A = area of the package (m2) x = thickness of the packaging material (mil)

p1 = partial pressure of O2 outside the package p2 = O2 inside

At steady state, both Equations (19) and (20) become equal as follows:

(21)

Equation (21) can be rewritten as follows:

= (Vc)(x)/(A)(c1 − c2) (22)

This equation suggests that the O2 permeability coefficient of a given MAP forfresh produce is a function of produce consumption rate of O2 (Vc), film thickness(x), area of the film (A) and the O2 concentration difference across the film (c1 − c2).

Hayakawa et al. (1975) developed a mathematical model for simulating gasexchange of packaged fresh produce and then obtained analytical solutions for the model.

PCO2

x---------- A pCO2i

pCO2O–( ) RCO2

W=

Vc c2( )∫=

Vd PO2( ) A( ) x 1–( ) p1 p2–( )=

PO2

Vc Vd c2( )∫ PO2( ) A( ) x 1–( ) p1 p2–( )= = =

PO2

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328 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

From these solutions, they derived simple algebraic formulae for the optimizationof packaging parameters.

(23)

(24)

(25)

(26)

whereyeqi = volumetric concentration of O2 in fresh produce package (atm) at

hypothetical equilibrium statezeqi = volumetric concentration of CO2 in fresh produce package (atm) at

hypothetical equilibrium statey = volumetric concentration of O2

z = volumetric concentration of CO2

yoi = volumetric concentration of O2 when τ = 0W = weight of fresh produce per one package (Kg)V = inside free volume of a package (cc)S = surface area of fresh produce package through which gas permeates

Ky = permeability of polymeric film to O2 (cc.mil/hr•in2•atm)

Kz = permeability of polymeric film to CO2 (cc.mil/hr•in2•atm)

ι = thickness of polymeric film (mil)t = time after packaging (hr)

Wei = lower quantity of CO2 from CO2 evolution rate curveWoi = weight when τ = 0

τ = t − ti : time after y becomes equal to yi or after z becomes equal to zi; (e.g., yi = the limit of (i − 1)th line segment for approximating a curve of Ry values (same for z)

f = constant used for approximating a curve for CO2 evolution rate of fresh produce

q = constant used for approximating a curve for O2 consumption rate of fresh produce

Equations (24) and (26) are similar to Equation (22) developed by Jurin andKarel (1963).

Deily and Rizvi (1981) integrated variables such as respiration rate, weight andoptimum gaseous composition requirements with packaging parameters such as per-meability, surface area and free volume into a set of analytical equations. These equa-tions can be solved to provide prediction of transient and equilibrium time valuesfor O2 and CO2 concentrations within a produce package or conversely, can be used

yeqi y–yeqi yoi–------------------- 1/V SKy /ι Woi+( )τ–[ ]exp=

yeqi yaky /ι qiw/s–( )/ ky/ι Woi/s+( )=

zeqi z–zeqi zoi–------------------- 1/V SKz /ι Woi+( )τ–[ ]exp=

zeqi zakz /ι fiw/s+( )/ kz/ι Wei/s+( )=

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Application of Packaging and Modified Atmosphere 329

to design a package that will help set up a known gaseous composition. The equationsare as follows:

y′ = ya − (W/SKy)Ry (27)

y(t) = y′ + (ya − y′)e−SKy t /V (28)

z′ = z + (W/SKz)Rz (29)

z(t) = z′ + (za − z′)e−SKz t /V (30)

Equation (27) can be rewritten as follows:

Ky = WRy/A(ya − y′) (31)

wherey′ = O2 concentration at hypothetical equilibrium state (atm)z′ = CO2 concentration at hypothetical equilibrium state (atm)ya = O2 concentration outside the package (atm) za = CO2 concentration outside the package (atm) z = volumetric concentration of CO2 gas inside fresh produce package (in atm)y = volumetric concentration of O2 gas inside fresh produce package (in atm)

Ky = O2 transmission rate of a polymeric film (cc of O2/m2•h•atm)

Kz = CO2 transmission rate of a polymeric film (cc of O2/m2•h•atm)

Ry = rate of consumption of O2 (cc/Kg)Rz = rate of evolution of CO2 (cc/kg)W = weight of fresh produce per one package (kg)S = surface area of produce-package through which O2 and CO2 can permeate

(m2)t = time after package (h)

V = inside free volume of a package (cc)

Equation (31) is likewise, similar to Equation (22) of Jurin and Karel (1963).The authors suggest that Equations (27) through (30) can be used to predict

equilibrium and transient state concentrations of O2 and CO2 at constant temperature.Since the time to reach equilibrium will be dependent upon the free volume insidethe package, they proposed that equilibrium concentration is independent of time,as indicated by Equations (27) and (29), even though the equations do not accountfor volume. In addition, they provided Equations (28) and (30) to calculate the timerequired for equilibrium to be reached.

Cameron (1990) calculated the rates of O2 uptake and CO2 production, assumingthat they equaled the rates of flux of the respective gases across the film as follows:

(32)

(33)

RO2PO2

A x 1– O2[ ]atm O2[ ]pkg–( )W 1–=

RCO2PCO2

A x 1– CO2[ ]pkg CO2[ ]atm–( )W 1–=

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330 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

These equations are again similar to Jurin and Karel’s (1963). For instance, Equa-tion (32) (oxygen uptake) can be rewritten as follows:

(34)

Cameron et al. (1989) developed mathematical equations describing O2 consumptionas a function of O2 concentration (for tomato fruit) and modeled film characteristics(O2 permeability coefficient, surface area, thickness) as a function of fruit weight.To predict film permeability characteristics for a sealed package containing a givenweight of fruit to yield a desired package O2 concentration, they developed thefollowing equation:

This equation is equivalent to the following:

(35)

This equation is again similar to Jurin and Karel’s (1963).Cameron et al. (1995) examined the factors that limit the ability to control gas

levels in MA packages. They suggested that while many researchers have recognizedthat gas partial pressures in the package atmosphere can be modeled, there are nocorrect models that use information on modifying atmosphere inside the plant tissue.Their solution was to model O2 uptake based on the Michaelis-Menton equation totake into account the enzymatic rate of O2 uptake in plant tissues. They suggestedthe following equation for estimating O2 permeability (Pi

film,T):

(36)

= the consumption rate of O2. The equation once rewritten as follows:

is identical to Jurin and Karel’s (1963) Equation (22).Talasila et al. (1995) developed a procedure to design MA packages for fresh

produce that accounts for changes in total pressure inside the package. Their modelis also affected by surrounding temperatures, product respiration and film perme-ability. They developed steady state equations to determine partial pressure of oxygenand carbon dioxide as follows:

(37)

(38)

PO2RO2x W/A O2[ ]atm O2[ ]pkg–( )=

PO2 A/∆x RO2

O2( )pkg[ ]W/ O2[ ]atm O2[ ]pkg–=

PO2∆x RO2

O2( )pkg[ ]W/A O2[ ]atm O2[ ]pkg–( )=

Pifilm,T ∆x W/Afilm( )RO2

max,T )[ ]/ O2( )ext O2( )pkg–[ ]=

RO2

Pi WR= O2 x/A O2( )ext O2( )pkg–[ ]

APMA/22.414E PA2 PA1–( )= RAW=

APMB/22.414E PB1 PB2–( )= RBW=

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Application of Packaging and Modified Atmosphere 331

where

A = surface area of the film (m2)PMA = oxygen permeation rateRAW = oxygen consumptionPA1 = atmospheric oxygen concentration (0.21 atm)PB1 = CO2 inside the packagePA2 = oxygen inside the packagePB2 = atmospheric CO2 (0.0 atm)

E = film thickness

Equations (37) and (38) can be written as follows:

which is again similar to Jurin and Karel’s (1963) Equation (22). They also suggestthat if the product respiration rate does not vary with time, the partial pressures ofgases inside the package at any given time are as follows:

(39)

(40)

These latter two equations also determine the times needed to reach any specific O2

and CO2 partial pressures.Exama et al. (1993b) suggest that in order to obtain the maximum benefit from

MAP systems, the steady state gas concentration should correspond to the storageoptima of the packaged commodity. They computed the film permeability requirementsby analyzing the kinetics of the MA process. Evolution of the volume fraction of O2

inside MA package (yiO2) as a function of time (t) was determined by the following:

(41)

Because at steady state, yiO2 remains constant [i.e., the left side of Equation (41) equalszero], and because it is desirable that at this state the internal O2 concentration is at theoptimum for the packaged produce (yiO2 = yoO2), Equation (41) can be rearrangedto give the required O2 permeability

(42)

so that

(43)

PMA RAW 22.44E/A PA2 PA1–( )=

PA1 t( ) PA1 t tss=( ) PA1 t tss=( ) PA1 t 0=( )–[ ] APMAGT1/22.414 EV1–( )t[ ]exp–=

PB1 t( ) PB1 t tss=( ) PB1 t tss=( ) PB1 t 0=( )–[ ]exp APMBGT1/22.414 EV1–( )t[ ]–=

dyiO2/dt APO2 p/VL( ) yeO2 yiO2–( )[ ] WRO2

/V( )–=

(PO2

R ):

APO2p/VL( ) yeO2 yoO2–( ) WRO2

/V=

PO2

R WRO2L/Ap yeO2 yoO2–( )=

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332 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Similarly, the permeation required to provide optimum CO2 concentration is as follows:

(44)

whereA = surface area of the film (m2)L = film thickness (mil)V = void volume inside the package (mL)

= O2 permeability of the filmyiO2 = volume fraction of O2 inside packageyeO2 = external O2 volume fraction yoO2 = internal optimum O2 volume fraction

W = weight of produce (Kg) = O2 consumption (respiration) rate = CO2 production rate

p = atmospheric pressure (1 atm)

Both Equations (43) and (44) are similar to Jurin and Karel’s (1963) Equation (22).Generally, all the models are based on mathematically combining respiration

and permeability characteristics of a given MAP/fresh produce system, and therefore,the outcomes are similar. However, this does not limit the power of such mathematicalmodels in facilitating material and product selection or package design in order tooptimize gas concentration through the shelf life of a MAP system. Most importantly,they represent a scientifically sound tool as opposed to a trial and error approach todevelop MAP applications. Due to the mathematical nature of such models, it ispossible to further advance the equations to include additional parameters and futuredevelopments in packaging materials or produce.

CASE STUDY OF FRESH-CUT APPLES

Gunes et al. (2001) studied cut ‘Red Delicious’ apple wedges stored at 5°C and treatedunder the following MA conditions: O2 (0.5, 1, 10, 21%) and CO2 (0, 7.5, 15, 30%).The samples that were treated with 30% CO2 and 0.5% O2 yielded the lowestfermentation products and ethylene production. The respiration rate measured forthose samples was = 2.1 Kg/hr and RQ = 1.9. According to Exama et al. (1993b),calculations of the required gas permeabilities for whole ‘Delicious’ apples, the dimen-sions of the packages used are as follows:

Area of the film (A) = 1320 cm2, film thickness = 1 mil, weight of apples =2.27 Kg, bulk volume of apples = 3818 cm3, container volume = 5090 cm3, andheadspace volume = 1272 cm3. Gas concentrations information is optimum CO2 con-centration is = 0.30, optimum O2 is = 0.005, external CO2 and O2 con-centrations are = 0, and = 0.21, respectively. Using Equations (43) and(44), the calculated required permeability for fresh-cut apples is as follows:

PCO2

R WRCO2L/Ap yoCO2 yeCO2–( )=

PO2

RO2

RCO2

RCO2

yoCO2yoO2

yeCO2yeO2

PCO2

R WRCO2L/Ap yoCO2 yeCO2–( ) =

2.27 Kg( ) 2.1 ml/Kg ⋅⋅⋅⋅ hr( ) 1 mil( )1320 cm2( ) 0.3 atm 0.0 atm–( )

-----------------------------------------------------------------------------------=

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Application of Packaging and Modified Atmosphere 333

= 9.228 × 10−3 mL mil/cm2 hr atm

Therefore, the permselectivity required for the product if stored under the studiedMA conditions would be

Figure 10.6 shows how permselectivity can be applied to cut fruit, ‘Red Deli-cious’ wedges in this case, given the recommended MA conditions (i.e., 30% CO2

and 0.5% O2). Figure 10.6, generated using the same approach as Figure 10.5, plots

FIGURE 10.6 Calculated optimum permselectivity for fresh-cut apple vs. fresh whole apple.

PCO2

R 0.012 mL mil/cm2 hr atm=

PO2

R WRO2L/Ap yeO2 yoO2–( ) =

2.27 Kg( ) 1.1 ml/Kg ⋅⋅⋅⋅ hr( ) 1 mil( )1320 cm2( ) 0.21 atm 0.005 atm–( )

------------------------------------------------------------------------------------=

PO2

R

PCO2

R /PO2

R 0.012 mL mil/cm2hr atm

9.228 10 3– mL mil/cm2hr atm×---------------------------------------------------------------------------=

PCO2

R /PO2

R 1.3=

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334 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

optimum atmospheres for both whole and cut apple wedges along with the calculatedpermselectivity required for cut apples (line 7). The permselectivities provided byseveral commonly available polymeric films are also plotted. None of the films pre-sented in the figure pass through the “cut apple” area, and thus, none perform satis-factorily to create the recommended MA for the cut apples (30% CO2 and 0.5% O2).Only the calculated (1.3) permselectivity line (7) passes through “cut apples.” Thissupports the theoretical findings discussed earlier and demonstrates the need todevelop films with lower β values than currently available polymers provide.

CONCLUSIONS

This example shows that the challenges facing the MAP of FCF cannot be overcomewithout considering the barrier properties of packaging material. Successful designand application of MAP for FCF must consider the CO2/O2 permselectivity as animportant property as gas permeability of a polymeric film. If gas permeabilitycoefficients can be viewed as a property that must match the respiration rate require-ment of fresh produce, then permselectivity must be viewed as a property that mustmatch the respiration quotient of the fresh produce. However, because most of theMAP application is used for fresh whole produce, the permselectivity of commonfilms may satisfy the required permselectivity with limited success that is not usuallydetected by the consumer. On the other hand, common films fail what is requiredby fresh-cut fruits, and therefore, rapid deterioration of the produce is evident.Therefore, material scientists and engineers must cooperate with plant physiologiststo innovate polymeric films that can meet and satisfy the permeability and CO2/O2

permselectivity required by FCF.

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Biotechnology and the Fresh-cut Produce Industry

Jennylynd A. James

CONTENTS

IntroductionApplications of Biotechnology in Agriculture

Historical Perspective and Conventional Plant BreedingDefinitions — Transgenic Crops and GMOsComparison of Traditional Plant Breedingand Genetic Modification of Crops

Commercially Available Genetically Modified CropsBiotechnology Applications in Fruit and Vegetable Production

Enhancement of Product Quality and Shelf LifeNutritional and Biomedical Benefits

Transgenic Plants as Vaccine Production SystemsEnvironmental BenefitsOther Beneficial Crops — Plants as Bioreactors

Food Safety ConcernsHuman Health and Environmental RisksSafety Assessment The U.S. PerspectiveAn International Perspective

Legal Considerations and LimitationsRegulation of GMOsLabeling of GMOs — The Debate

ConclusionsReferences

INTRODUCTION

Biotechnology can be defined as the manipulation of biological systems, livingorganisms or their derivatives to make products or modify processes for a determinedend use. This technology has been implemented for centuries with the discovery offermentation techniques and plant breeding by ancient civilizations. In its modern

11

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form, biotechnology has assumed new methods of altering genetic information oforganisms, the most significant of these being genetic engineering. The tools ofbiotechnology have been used in recent times in food processing and medicine toproduce enzymes at commercial levels. Biotechnology has enhanced life and willcontinue to do so by providing useful tools for human health and nutrition.

Plant breeding has been conducted by many cultures over centuries to improvecrop yields and produce varieties of crops with new and improved qualities. It isonly within the last two decades, with the development of biotechnology tools, thatnew technologies have been included in plant breeding. Scientists have found a wayto transfer genes coding for desired characteristics into plants to produce viable newcrops. Most of the success has been achieved in sustainable agricultural crops likecorn, wheat and other grain. In recent years, scientists have been investigating waysto enhance sensory qualities of perishable crops like fruits and vegetables, to extendshelf life for the fresh market and processing and to improve color, flavor and otherqualities. The FLAVR SAVR

TM

tomato was the first fresh produce item to getattention for commercial production (Calgene, Inc., Davis, California).

The use of biotechnology to improve the food supply has had mixed reactionsworldwide. It is seen by some as a fast and efficient improvement on traditionalbreeding techniques. Purists have discredited the technology saying that scientistsare tampering with nature. Several methods are used to identify genetically mod-ified organisms (GMOs) including polymerase chain reaction (PCR) technologyand enzyme-linked immunosorbent assay (ELISA). Before GMOs can be com-mercialized, they are supposed to be tested extensively to ensure that no harmfuleffects have been introduced and the new product is “substantially equivalent” tothe old. Are safety assessment methods thorough and statistically sound or are themethods outdated and in need of revision as new technologies are introduced?There is no sufficient data available to disprove long-term damage to humans or theenvironment.

The fear of the unknown has triggered hysteria in some regions of the world.Trade barriers are being instituted to prevent entry of genetically modified foods.Calls are being made to growers and manufacturing operations who use GMOs tolabel products as “Genetically Modified.” Labeling of a product is thought to be oneway of giving consumers the opportunity to choose and be informed. Labeling is,however, very expensive to implement, and the benefits of labels have been disputedby many in the produce industry. The labeling of a GMO may have negativeconnotation for a crop that is perfectly sound. Also, new laws are being formulatedby government regulators to encompass GMOs that were not, until recently, givenany separate regulatory attention, apart from any new food being introduced. Nouniformity in regulation yet exists worldwide. Thus, future trade will become moreand more complicated.

This review seeks to focus on some of the prominent applications of agricul-tural biotechnology. Mention is made of the work done to develop new crops andof the laws and regulations used as guidelines in the production of geneticallyengineered foods. The possibilities of agricultural biotechnology are limited onlyby one’s imagination. It is hoped that this technology will not be overregulated

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so as to stifle the production of ingenious food products or the solution of problemsfaced in the food industry and agriculture. Because human subjects cannot be usedfor safety assessment studies, as with other new technologies, only time will tellthe effects.

APPLICATIONS OF BIOTECHNOLOGY IN AGRICULTURE

H

ISTORICAL

P

ERSPECTIVE

AND

C

ONVENTIONAL

P

LANT

B

REEDING

Agricultural biotechnology in the fundamental sense could be traced to humanactivity for thousands of years. Traditional breeding methods have introduced sub-stantial improvements to generate crops that would not otherwise occur in nature.One successful example of the use of traditional breeding methods that evolvedthrough history was the work done by Native Americans over 8000 years to developmodern corn from a wild plant called teosinte. The ancient ancestor of corn wassmall and did not possess the large, succulent grain of the modern variety. However,through years of crossbreeding and selection of viable plants that bore these largegrains, the modern variety evolved into what we know today. The history of agricul-tural biotechnology is summarized in Table 11.1 (IFT Expert Committee, 2000a,c).

The origin of the concept of inheritance may be traced back to the times ofancient Greece when Theophrastus, a student of Aristotle, first recognized the anal-ogy between animal and plant reproduction and coined the words male and femaleto describe the participants in sexual reproduction. However, the concept of a genebegan with Gregor Mendel in the 1860s, although the word itself was not formeduntil scientists repeated and extended Mendel’s work during the early twentiethcentury. The term “gene” was introduced by W. Johannsen in 1910 and referred toa hypothetical unit of information that determines the inheritance of an individualcharacteristic in an organism. The existence of genes was inferred from the statisticaldistribution of simple heritable traits as studied by Mendel in plant breeding exper-iments. The Mendelian view of inheritance in eukaryotes was defined by the occur-rence of independent segregation and the independent assortment of different allelicpairs of genes, each specifying a different trait.

Crossbreeding methods are used to transfer the complete set of genes from theparent plants to the new offspring. Not only are one or two desired genes introduced,but thousands of other genes, some benign and some undesirable, are also exchanged.Expensive, time-consuming methods are used to remove unwanted genes and toretain the desired improvements. One conventional breeding method used to reshufflegenes is the production of “double-cross hybrids.” The breeder crosses a variety Xwith Y to produce Z, variety A is crossed with B to make C, then C and Z are crossedto produce the desired seed. Another conventional breeding method that is widelyaccepted is “mutation breeding,” in which ionizing radiation or other mutagenicagents are used to create genetic changes. Conventional breeding methods are limitedin that if a desired gene is not available or cannot be mutated from an existing gene,then the desired trait cannot be achieved (McHughen, 2000).

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Organisms have been genetically engineered to overproduce bioingredients andimprove food fermentations, especially in the making of wine, beer and cheese.Genetic engineering has also been used in the detection of food pathogens, usingDNA probes and monoclonal antibodies, and for the effective utilization of food-processing wastes. Recombinant DNA technology has provided powerful and novelapproaches to understanding the complex mechanisms by which eukaryotic geneexpression is regulated, and this technology could provide countless benefits andimprovements in food production.

TABLE 11.1History of Agricultural Biotechnology and Recent Applications of New Technologies

Date Discovery

2000 BC Cultivation19th Century Selective crossbreeding (in Europe—Gregor Mendel) Early 20th Century Mutagenesis and selectionMid 20th Century Cell culture1930s Somoclonal variation1940s Embryo rescue1950s Polyembryogenesis1970s Anther culture1970 Norman Borlaug, first plant breeder to win a Nobel Prize for his work in

Green Revolution wheat varieties (high yield)1980 Recombinant DNA1980s Marker-assisted selection1980s and 1990s Chloroplast transformation to increase transformation efficiency and control

gene flowCobombardment using the gene gun to add multiple genes simultaneouslyTransposon tagging, positive selection exclusive energy sources, an effective alternative to antibiotic selection.

Targeted site-specific recombination to target gene inserts to specific sites in plant tissue

Chimeraplasty to create subtle alterations in the plant’s own genes, for example, to produce herbicide tolerance without introducing novel genes

1990s Genomics1994 The FLAVR SAVR

TM

tomato, first product of agricultural biotechnology approved for U.S. grocery stores

1995 Soybeans, products of agricultural biotechnology, introduced on the market1997 U.S. government accepted 18 crop applications of biotechnology1999 Development of ‘Golden Rice’ which contained beta-carotene, a precursor

of vitamin A2000 and beyond Bioinformatics

Designing plants for herbicide tolerance, insect protection, disease protection, improved nutrition profiles

Source:

Adapted from McGloughlin (2000) and Council for Biotechnology Information (2000).

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D

EFINITIONS

— T

RANSGENIC

C

ROPS

AND

GMO

S

Transgenic crops are those plants that are derived using recombinant DNA technol-ogy and other biotechnologies to form a new variety that expresses the trait codedby the inserted gene(s). Recombinant DNA methods enable breeders to select,transfer or modify single genes thereby eliminating the need to “select out” unde-sirable genes. R-DNA technology allows the insertion of useful genes from anyother species. Genetically modified (GM) refers to the fact that the plant genomehas been altered by the addition or removal of a gene. In order to successfully transfera gene or genes coding for a specific trait, the plant genome should be sequencedand the gene function elucidated, an area of study called genomics. There is func-tional genomics that studies specific traits from gene codes, as well as structuralgenomics that includes the genetic mapping, physical mapping and sequencing ofentire genomes. Many databases already exist to distribute molecular information.However, the post-genomic era will require many more to collect, manage andpublish the influx of new research results. The future of agricultural biotechnologywill depend in part on advances in sequencing, genome analysis and informationtechnology to characterize beneficial genes for crop improvement.

C

OMPARISON

OF

T

RADITIONAL

P

LANT

B

REEDING

AND

G

ENETIC

M

ODIFICATION

OF

C

ROPS

Traditional biotechnology has been used for thousands of years, since the advent ofthe first agricultural practices for the improvement of plants, animals and microbes.Selective breeding was used to exchange genetic information between two relatedplant parents, producing progeny which had desired properties, for example,increased yields and improved taste. Traditional breeding, however, requires that thetwo plants being crossed be closely related or of the same species. Thus, active plantbreeding has led to the development of superior plant varieties over centuries, farmore rapidly than random mating in the wild. Traditional breeding is, however, time-consuming and many times the characteristic of interest does not occur in a relatedspecies. This is where modern biotechnology methods have assisted traditionalbreeding methods.

Some scientists consider genetic engineering an extension of conventional breed-ing, while others hold the view that it differs profoundly. Conventional breedingdevelops a new plant variety by the process of selection, and genetic material thatis already present within a species is expressed. The exception to this would behybridization, wide crosses and horizontal gene transfer (Hansen, 2000). Geneticengineering allows the insertion of a gene, and this must be followed up by selection.A promoter gene from a virus is usually inserted to make the new gene expressitself. This whole process is significantly different from conventional breeding, evenif the goal is to insert genetic material from the same species. There is an increase inprecision when the gene carrying the trait of interest is known. Also using geneticengineering techniques, the potential sources from which desirable traits may beobtained are increased. The entire span of genetic capabilities available among allbiological organisms can potentially be used in any other organism.

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There are major differences in the success rates of conventional breeding vs.use of genetic engineering. In nature, most offspring are viable, and in conventionalbreeding, scientists grow many plants and keep only a few with the most desirabletraits. In the early development of genetic engineering, although cells containing theinserted gene of interest were selected, it was still necessary to grow whole plantsfrom these cells to determine whether the gene was expressed giving the desiredtrait. A large percent of engineered cells were not viable or failed to produce thedesired trait. In successive plant generations, only one in thousands (or millions) ofcells is able to incorporate the desired trait and express this for generations withoutproducing undesirable side effects. This indicates that genetic engineering is not asefficient as it is advertised to be (Hansen, 2000; Walden and Wingender, 1995).

Genetic engineering controls the trait that is to be introduced, yet cannot controlthe location of introduction. Traditional breeding, however, occurs between organ-isms that share a recent evolutionary history, so shuffling occurs around alleles,different versions of the same gene. These genes are usually fixed in location on thechromosome by evolution. A foreign promoter, usually from a plant virus, is usedto enhance the expression of transgenes in genetic engineering, and the introductionof foreign DNA is not used in traditional breeding (Meyer, 1995).

Figure 11.1 shows a schematic comparison of conventional breeding methods vs.genetic engineering techniques. The precision of transforming DNA with the geneof interest eliminates the need to do a series of back crossing to remove unwantedproperties in traditional breeding methods. Table 11.2 summarizes the differencesbetween conventional breeding and genetic engineering. One of the main technical

FIGURE 11.1

Classical plant breeding vs. genetic engineering. Traditional plant breedingcombines many genes at one time, while in plant biotechnology, a single gene may be addedto the commercial variety.

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limitations in the improvement of plants using r-DNA technology is that there is notenough genomic data for all commercially produced crops. The DNA sequences ofall plants must be studied to identify the location and function of all genes. Experi-ments have shown that many sequences are conserved among species, and the samegene confers the same trait in different species (Persley and Siedow, 1999).

There are, thus, key scientific differences between genetic engineering and con-ventional breeding, in terms of the process and the genetic makeup of the product.Recoverable DNA from genetically engineered plants would usually reveal a viralpromoter, genetic material from

Agrobacterium

and, in many cases, a bacterial anti-biotic marker gene. These are never deliberately introduced in conventional breedingof plants.

C

OMMERCIALLY

A

VAILABLE

G

ENETICALLY

M

ODIFIED

C

ROPS

Agricultural biotechnology has been applied in the improvement of agronomic andquality traits, as well as the production of novel crop products and renewableresources. This technology has been used to develop crops with pesticide resistance,improved yield, ability to use marginalized land, improved nutritional benefits,reduced environmental impact and pharmaceutical benefits like vaccines (ThirdWorld Academy of Sciences, 2000). Early products of agricultural biotechnologyfocused mainly on agronomic traits like those related to biotic stress: insect resis-tance, disease resistance (viral, bacteria, fungal, nematode) and weed-herbicidetolerance. Work has been done to provide relief from abiotic stresses like drought,cold, heat and poor soils, and to improve yields by nitrogen assimilation, starchbiosynthesis and oxygen assimilation (Wilkinson, 1997).

Research to improve quality traits has been developed in the areas of processingproperties: extension of shelf life; altering reproductive methods by creating sexbarriers, male sterility and seedlessness; production of nutraceutical plants withimproved protein, carbohydrates, fats, vitamins, etc.; and plants with the ability to

TABLE 11.2Classical Breeding vs. Genetic Engineering

Classical Breeding Genetic Engineering

(A) Use living plant machinery Use plant machinery in the laboratory(B) Random genetic exchange using entire genomes of

both plantsThere is an exchange of a specific gene

or genes(C) Breeding must occur between closely related

speciesGenes from plants or other organism may

be used to transform the plant(D) Control of gene expression depends on source of

genetic materialWhen and where the gene is expressed

can be controlled(E) Inclusion of ancillary, unwanted traits that must be

eliminated by generations of back crossingPrecise traits are transferred

(F) Progress is lengthy If the gene coding for a trait is known, there is rapid development of varieties with new and desirable traits

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remove toxins and phytase. Improvements in taste, architecture, fiber content andin ornamentals, changing color, shelf life, morphology and fragrance are all qualitytraits geared for improvement using biotechnology. Novel crop products like oilswith special properties, proteins and polymer production, as well as vaccine pro-duction in plants, are some of the future applications of agricultural biotechnology.

Products found initially on the market would not be specialty items but stapleslike flour, meal or oil extracted from genetically modified crops. Commercial pro-duction of novel transgenic crops first focused on agronomic benefits like increasedproductivity of crops with reduction of production by decreasing the need for inputsof pesticides. This has been studied mostly in crops grown in temperate zones. Theintense and expensive research and development of transgenic plants over twodecades has led to the commercial production of new varieties over the last fouryears (Persley, 1999).

Herbicide resistance has allowed the possibility of reducing chemical applicationof herbicides during large-scale farming. The application of agricultural biotechnol-ogy could mean an improvement in the quality of life, because new strains of plants,giving higher yields with fewer inputs, will be grown in a broader range of envi-ronments. Natural resources will be conserved, providing more nutritious productswith a longer shelf life at an economical cost to consumers. Multiple benefits for growersinclude more flexibility in terms of crop management (especially for herbicide-tolerant crops), decreased dependency on conventional insecticides and herbicides,higher yields and cleaner and higher grades of grain/end products (Vasil et al., 1992).

Commercial products that have been enhanced using biotechnology can be foundin Table 11.3. Preliminary work focused on large-volume, sustainable agriculturalcrops like corn, soybeans and potatoes that require fewer applications of herbicides.Biotechnology-enhanced soybeans have been created with a lower saturated fat con-tent and higher oleic acid content, thus offering better frying stability of soya beanoil without further processing. ‘Roundup Ready’ soybean is one example of a trans-genic crop with herbicide tolerance. U.S. farmers are said to have saved an estimated$330 million in 1998 because of lower herbicide costs in growing “Roundup Ready”soybeans. There was a reduction in crop injury and an improvement in weed control.The application also encourages adoption of no-till, which saves the environmentfrom erosion. Herbicide-tolerant crops also benefit water quality. “Roundup Ready”corn was planted at five Illinois watersheds in 1999. All sheds had past problemsof atrazine in excess of 50 ppb, however, samples collected in 1999 were above the3 ppb standard for the five watersheds using “Roundup Ready” corn (McGloughlin,2000). The U.S. farmer benefited with 76% of the $100 million saved in 1997 using“Roundup Ready” soybean. The consumer benefited only 4% (Falck-Zepeda et al.,1999).

The problems of the Colorado potato beetle (CPB) and corn rootworms (CRWs)caused billions of dollars in losses to farmers in the United States in the past becauseof loss of crops and increased pesticide application costs. Thus, using agriculturalbiotechnology to create insect resistance in crops was a very exciting prospect forfarmers (NRC, 2000a; Vaek et al., 1987). The first insect-resistant crops were tobaccoand tomato plants that used a native truncated lepidopteran-specific insect toxin genefrom

Bacillus thuringiensis

subsp.

kurstaki

(Btk). The field testing conducted in

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TABLE 11.3Beneficial Crops Derived Using Biotechnology

Year/Firm New Variety Trait Gene and Source

2000

Aventis Male-sterile corn The barnase gene from

Bacillus amyloliquefaciens

1999

Agritope Inc. Modified fruit-ripening cantaloupe

S-adenosylmethionine hydrolase gene from

Escherichia coli

bacteriophage T3BASF AG Phytaseed canola The phytase gene from

Aspergillus niger

var. van

Tieghem

Rhone-Poulenc Ag. Co.

Bromynil-tolerant canola The nitrase gene from

Klesiella pneumoniae

subsp.

ozaenae1998

AgrEvo, Inc. Glufosinate-tolerant soybean

Phosphinothricin acetyltransferae gene from

Streptomyces viridochromogenes

Glufosinate-tolerant sugar beet

Phosphinothricin acteyltransferase gene from

S. viridochromogenes

Insect-protected and glufosinate-tolerant corn

The cry9C gene from

Bacillus thuringiensis

(Bt) subsp.

tolworthi

and the bar gene from

Streptomyces hygroscopicus

Male-sterile or fertility-restorer and glufosinate-tolerant canola

The male-sterile canola contains the barnase gene, and the fertility-restorer canola contains the barstar gene from

B. amyloliquefaciens,

both lines have the phosphinothricin acetyltransferase gene from

S. viridochromogenes

Calgene, Inc. Bromoxynil-tolerant/insect-protected cotton

Nitrilase gene from

Klebsiella pneumoniae

and the cryIA(c) gene from

B. thuringiensis

subsp.

kurstaki

Insect-protected tomato The cryIA(c) gene from

B. thuringiensis

subsp.

kurstaki

Monsanto Co. Glyphosate-tolerant corn A modified enolpyruvilshikimate-3-phosphate synthase gene from corn

Insect- and virus-protected potato

The cryIIIA gene from

B. thuringiensis

sp.

tenebrionis

and the Potato Leaf Roll Virus replicase gene

Insect- and virus-protected potato

The cryIIIA gene from

B. thuringiensis

sp.

tenebrionis

and the Potato Virus Y coat protein geneMonsanto Co./Novartis

Glyphosate-tolerant sugar beet

The enolpyruvylshikimate-3-phosphate synthase gene from

Agrobacterium

sp. strain CP4, and a truncated glyphosphate oxidoreductase gene from

Ochrobactrum anthropi

Pioneer Hi-Bred Male-sterile corn The DNA adenine methylase gene from

E. coli

University of Saskatchewan

Sulfonylurea-tolerant flax Acetolactase synthase gene from

Arabidopsis

1997

AgrEvo, Inc. Glufosinate-tolerant canola

Phospinothricin acetyltransferase gene from

S. viridochromogenes

(

continued

)

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TABLE 11.3 Beneficial Crops Derived Using Biotechnology (Continued)

Year/Firm New Variety Trait Gene and Source

Bejo Zaden BV Male-sterile radichio rosso

The barnase gene from

B. amyloliquefaciens

Dekalb Genetics Corp.

Insect-protected corn The cryIA(c) gene from

B. thuringiensis

DuPont High-oleic-acid soybean Sense suppression of the GmFad2-1 gene which encodes a delta-12 desaturase enzyme

Seminis Vegetable Seeds

Virus-resistant squash Coat protein genes of Cucumber Mosaic Virus Zucchini Yellow Mosaic Virus and Watermelon Mosaic Virus

University of Hawaii/Cornell University

Virus-resistant papaya Coat protein gene of the Papaya Rinspot Virus

1996

Agritope Inc. Modified fruit ripening tomato

S-adenosylmethionine hydrolase gene from

E. coli

bacteriophage T3

Dekalb Genetic Corp.

Glufosinate-tolerant corn Phosphinothricin acetyl transferase gene from

S. hygroscopicus

DuPont Sulfonylurea-tolerant cotton

Acetolactate synthase gene from tobacco,

Nicotiana tabacum

cv.

Xanthi

Monsanto Co. Insect-protected potato The cryIIIA gene from

B. thuringiensis

Insect-protected corn The cryIA(b) gene from

B. thuringiensis

subsp.

kurstaki

Insect-protected corn The cryIA(b) gene from

B. thuringiensis

subsp.

kurstaki

Northrup King Co. Glyphosate-tolerant/insect-protected corn

The endopyruvylshikimate-3-phosphate synthase gene from

Agrobacterium

sp. strain CP4 and the glyphosphate oxidoreductase gene from

O. anthropi

in the glyphosate tolerant lines; the cryIA(b) gene from

B. thuringiensis

subsp.

kurstaki

in lines that are also insect protectedPlant Genetic Systems NV

Insect-protected corn The cryIA(b) gene from

B. thuringiensis

subsp.

kurstaki

Male-sterile and fertility-restorer oilseed rape

The male-sterile oilseed rape contains the barnase gene from

B. amyloliquefaciens

, the fertility-restorer lines express the barstar gene from

B. amyloliquifaciens

Male-sterile corn The barnase gene from

B. amyloliquifaciens

1995

AgrEvo Inc. Glufosinate-tolerant canola

Phosphinothricin acetyltransferase gene from

S. viridochromogenes

Glufosinate-tolerant corn Phosphinothricin acetyltransferase gene from

S. viridochromogenes

Calgene, Inc. Laurate canola The 12:0 acyl carrier protein thioesterase gene from California bay,

Umbellularia californica

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1987 and 1988 showed a reduction in damage caused by the tomato fruit worm.However, the expression of the Bt protein was too low for commercial use. Aredesigned synthetic Bt gene was created with a 500-fold increase in expression ascompared to the wild type. Transgenic potato plants expressing the synthetic genefrom

B. thuringiensis

subsp.

tenebrionis

at high levels exhibited strong resistanceto CPB in a large number of field trials. This new potato crop introduced in the mid1990s significantly reduced the use of undesirable insecticides in the environment,as well as saved farmers millions in insecticide application (Shah et al., 1995).

Insect damage was also a serious problem for cotton crops with lepidopteraninsects such as cotton bollworm, being responsible for US $216 million in losses inthe mid 1990s. Genetically modified cotton plants with an agronomically usefullevel of resistance to ballworm were developed through the expression of syntheticBtk gene at high levels. Also, the European corn borer (ECB) was a major corn pestin North America and Europe, causing loss in yield of 3–7% annually (Shah et al.,1995). The economic losses in Illinois alone amounted to US $50 million in 1995.

TABLE 11.3 Beneficial Crops Derived Using Biotechnology (Continued)

Year/Firm New Variety Trait Gene and Source

Ciba-Geigy Corp. Insect-protected corn The cryIA(b) gene from

B. thuringiensis

subsp.

kurstaki

Monsanto Co. Glyphosate-tolerant corn Enolpyruvylshikimate-3-phosphate synthase gene from

Agrobacterium

sp. strain CP4Glyphosate-tolerant canola

Enolpyruvylshikimate-3-phosphate synthase gene from

Agrobacterium

sp. strain CP4Insect-protected cotton The cryIA(c) gene from

B. thuringiensis

subsp.

kurstaki

1994

Asgrow Seed Co. Virus-resistant squash Coat protein genes of Watermelon Mosaic Virus 2 and Zucchini Yellow Mosaic Virus

Calgene, Inc. FLAVR SAVR

TM

tomato Antisense polygalacturonase gene from tomatoDNA Plant Technology Corp.

Bromoxynil-tolerant cotton

A nitrilase gene isolated from

Klebsiella ozaenae

Monsanto Co. Improved-ripening tomato A fragment of the aminocyclopropane carboxylic acid synthase gene from tomato

Zeneca Plant Science

Glyphosate-tolerant soybean

Enolpyruvylshikimate-3-phosphate synthase gene from

Agrobacterium

sp. strain CP4Improved-ripening tomato Aminocyclopropane carboxylic acid deaminase

gene from

Pseudomonas chloraphis

strain 6G5Insect-protected potato The cryIIIA gene from

B. thuringiensis

sp.

tenebrionis

Delayed-softening tomato A fragment of the polygalacturonase gene from tomato

Source:

Reprinted with permission from the Institute of Food Technologists (IFT Expert Committee,2000c).

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A synthetic Btk gene was introduced into corn to provide resistance to ECB. Com-mercialization of high-valued, insect-resistant crops decreased the use of chemicalinsecticides and chemical residues in the environment. There was the concern,however, that the use of Bt proteins for the control of insects would develop insectresistance to Bt toxins. Thus, resistance-management strategies are combined withintegrated pest management (IPM) procedures. Some strategies include using thehigh-dose expression of Bt genes, using host plants for sensitive insects (Koziel et al.,1993, 1996). Also, agronomic practices that minimize insect exposure to Bt and anIPM system are important. Long-term strategies use the multiple genes encodinginsect-control proteins with unique modes of action in the same crop. Research hasbeen conducted to find non-Bt proteins to control insect pests. Examples of insec-ticidal plant proteins include lectins, amylase inhibitors and protease inhibitors thatcan retard insect growth and development when ingested at high levels. Thesecompounds, however, do not provide the same mortality rate of Bt.

An indirect benefit of cultivating Bt corn is a decreased risk of low grain qualityby mycotoxins. Research showed that ears and grain from ‘YieldGard’ corn were lesscontaminated by fusarium and fumonisin than conventional corn. Bt crops hadimproved insect control, improved farm efficiency, reduced crop injury with improvedquality and reduced insecticide exposure. A large percent of the benefits (42%) ofgrowing Bt corn accrued to the farmer in 1997. In 1998, 22 of the 75 million acresof corn planted were Bt corn. In a November 1999 Iowa State study, 26% of thefarmers planting Bt corn were able to reduce their pesticide use, and there were at least6 million acres of land with little or no pesticide application [International Food Infor-mation Council (IFIC), 1999]. Also in 1999, adopters of genetically engineered corn,soybeans and cotton combined used 7.6 million fewer acre treatments (2.5%) ofpesticide than nonadopters in 1997 (Falck-Zepeda et al., 1999). Fewer pounds ofinsecticide (2 million) were used in 1998 to control bollworm and budworm in 1998than in 1995, before the introduction of Bt cotton. Cotton farmers increased theiryields by 85 million pounds and made $92 million more than those who did not adoptthe new technology (McGloughlin, 2000).

Annual losses of over $100 billion are incurred because of nematode damageto crops (Atkinson et al., 1995). Plant-parasitic nematodes are quite numerous andhave adopted subtle yet damaging interactions with host plants. Nematicides areused to control these pests, but in many cases, these hazardous chemicals are noteffective. Agricultural technology may offer one means of controlling nematodes.One approach requires promoters that direct a specific pattern for genes encodingeffector proteins. Effectors may act directly against the nematode or disrupt modi-fication of the plant cell by the parasite (Atkinson et al., 1995).

Seed companies and produce growers have taken on the challenge of usingagricultural biotechnology to improve crops in the United States, and according toU.S. Secretary of State, Dan Glickman, more than 70 million acres of geneticallymodified crops were grown in 1999. Field corn and soybeans make up the vastmajority of agricultural biotech products. However, sweet corn, squash and potatoesare also being produced (Shee, 2000).

In terms of international growth, in 1999, an estimated 40 million hectares of landworldwide, were cultivated with transgenic plants of over 20 species, the most important

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being cotton, corn, soybean and rapeseed (James, 1999). Summaries of the global areaof transgenic crops, the types of crops commercialized and the new traits promotedin transgenic crops, are contained in Tables 11.4, 11.5 and 11.6. Twelve countries,eight industrial and four developing, have contributed to a more than 20-fold (×23.5)increase in the global area of transgenic crops between 1996 and 1999. High adoptionrates of new transgenic crops reflect grower satisfaction with the products that offersignificant benefits ranging from more convenient and flexible crop management,

TABLE 11.4Global Area of Transgenic Crops in 1998 and 1999: By Crop (Millions of Hectares)

Crop 1998 % 1999 % Increase (Ratio)Soybean 14.5 52 21.6 54 7.1 (0.5)Corn 8.3 30 11.1 28 2.8 (0.3)Cotton 2.5 9 3.7 9 1.2 (0.5)Canola 2.4 9 3.4 9 1.0 (0.4)Potato <0.1 <1 <0.1 <1 <0.1 (—)Squash 0.0 0 <0.1 <1 (—) (—)Papaya 0.0 0 <0.1 <1 (—) (—)

Total 27.8 100 39.9 100 12.1 (0.4)

Source: James (1999), ISAAA.

TABLE 11.5Global Area of Transgenic Crops in 1998 and 1999: By Country (Millions of Hectares)

Country 1998 % 1999 %Increase 1998 to

1999 (Ratio)Argentina 4.3 15 6.7 17 2.4 (0.6)Canada 2.8 10 4.0 10 1.2 (0.4)China <0.1 <1 0.3 1 0.2 (3.0)Australia 0.1 1 0.1 <1 <0.1 (—)South Africa <0.1 <1 0.1 <1 <0.1 (—)Mexico <0.1 <1 <0.1 <1 <0.1 (—)Spain <0.1 <1 <0.1 <1 <0.1 (—)France <0.1 <1 <0.1 <1 <0.1 (—)Portugal 0.0 0 <0.1 <1 <0.1 (—)Romania 0.0 0 <0.1 <1 <0.1 (—)Ukraine 0.0 0 <0.1 <1 <0.1 (—)United States 20.5 74 28.7 72 8.2 (0.4)

Total 27.8 100 39.9 100 12.1 (0.4)

Source: James (1999), ISAAA.

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352 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

higher productivity or net returns/hectare and a safer environment through decreaseduse of conventional pesticides, leading to more sustainable agriculture (James, 1999).

Seven major transgenic crops were commercialized in 1999. They were soybean,corn/maize, cotton, canola/rapeseed, potato, squash and papaya (Table 11.4). Thetraits of significant importance were herbicide tolerance and insect resistance. Therewas also an increase in production of crops with virus resistance traits in 1999,mainly potatoes, squash and papaya, less than 1% annual acreage. The global marketfor transgenic crops has grown rapidly over five years, from $75 million in sales in1995 to an estimated $3 billion in sales in the year 2000. This is projected to increaseto $25 billion in 2010 (James, 1999). The global production of transgenic cropsincreased from 4.3 million acres in 1996 to 98.6 million acres in 1999. The UnitedStates accounted for 72% of the global area, followed by Argentina (17%), Canada(10%), China (1%) and Australia and South Africa (0.23 million acres).

As agricultural biotechnology applications increase, there will be a change of focusfrom beneficial profiles for growers to benefits for consumers in the high-value-addedmarkets. The pace of biotechnology-driven consolidations in industry was slower in1999 than in the previous three years. Most of the large multinational companies withinvestments in seeds, crop biotechnology and crop protection are restructuring anddownsizing programs, and this could lead to new alliances and mergers. A globalreview of commercialized transgenic crops was written by James (1999).

BIOTECHNOLOGY APPLICATIONS IN FRUIT AND VEGETABLE PRODUCTION

As expansion of transgenic crops continues, there will be a shift from the currentgeneration of “input” agronomic traits to the next generation of “output” quality traits,which will result in improved and specialized nutritional food and feed products tosatisfy the high-value-added market. This movement will broaden the beneficiary profilefrom growers to consumers, perhaps improving public acceptance (James, 1999).

Enhancement of Product Quality and Shelf Life

Improvements targeted in the fresh fruits and vegetables and fresh-cut industry includeimproved sweetness in peas and producing higher crop yields, smaller seedless melons

TABLE 11.6Global Area of Transgenic Crops in 1998 and 1999: By Trait (Millions of Hectares)

Trait 1998 % 1999 % Increase (Ratio)Herbicide tolerance 19.8 71 28.1 71 8.3 (0.4)Insect resistance (Bt) 7.7 28 8.9 22 1.2 (0.2)Bt/herbicide tolerance 0.3 1 2.9 7 2.6 (8.7)Virus resistance/other <0.1 <1 <0.1 <1 <0.1 (—)

Global totals 27.8 100 39.9 100 12.1 (0.4)

Source: James (1999), ISAAA.

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Biotechnology and the Fresh-cut Produce Industry 353

for use as single servings, delayed ripening in bananas and pineapples and fungalresistance in bananas. Recent successes in enhancing fruits and vegetables for the freshfruit and processing markets include the biotechnological modification of tomatoes tosoften slower and remain on the vine longer, resulting in more flavor and color. Tomatoesare one of the most important world crops in terms of sales and their overall contributionto nutrition. Tomatoes are used either fresh or for processing, and thus, distinct qualitiesare required for the different markets. Fresh tomatoes should have an acceptable flavorand firm structure, while processing tomatoes require suitable rheological characteristicsfor production of juice, ketchup and other products. A systematic approach was under-taken to understanding tomato ripening at the molecular level, and ripening was con-trolled to improve quality and reduce postharvest losses. Two methods to achieve thiswere by altering the activity of cell wall enzymes that are involved in softening orblocking the biosynthesis of ethylene, the fruit-ripening hormone (Schuch, 1994).

Scientists at Zeneca Plant Science in Germany were able to study the hormonesresponsible for the rate of ripening and the biosynthesis and deposition of carotenoidsin chromoplasts determining color pigment production. They also studied the metab-olism of sugars and acids involved in flavor development and factors affecting fruitfirmness in modifying the structure and composition of cell walls. Genes controllingthese quality parameters were identified. Polygalacturonase, which hydrolyzes α-1,4linkages in the polygalacturonic (PG) acid component of cell walls, and pectinesterase(PE) were identified as major cell-wall-modifying enzymes formed during tomatoripening. The plant genome was modified to introduce genes in an antisense orientation(Smith et al., 1988, 1990), thus reducing levels of these enzymes in ripened tomatoes.Genetically modified tomatoes had improved processing characteristics for Bostwickviscosity, serum consistency and soluble solids content. The fresh market attributes oflow PG tomatoes led to improved postharvest handling (Kramer et al., 1992; Schuchet al., 1992; Gray et al., 1993). They were able to last on the vines for a longer time,thus developing more flavor. Calgene, a California-based biotechnology company,developed and commercialized a product called the FLAVR SAVRTM tomato for thefresh market using this technology. The product was not commercially successful,however, mainly because of problems in marketing and ability to supply large volumes.

Tomatoes have also been cloned with disease resistance genes. The Pto gene thatconfers resistance to bacterial speck has been cloned from wild tomato species. Pro-grammed cell death in plants (apoptosis) has been studied, elucidating the pathwayslinking PCD to disease, expression of novel genes or signal molecules to alter pathogeninvasion or symptom expression. Tomato plant resistance to the fungus Alternariaalternata sp. lycopersici was achieved by the transformation of tomato plants with aninhibitor of PCD, the baculovirus p35 gene. Genetically modified plants thrived in thepresence of the fungus, compared to the susceptible wild type. Lesions on inoculatedfruit were much smaller. Also, the p35 expressing plant had increased resistance tobacteria, showing decreased infection in response to infection by Pseudomonas syrin-gae pv. tomato and Colletotrichum coccodes (McGloughlin, 2000).

Another example of consumer benefits from agricultural biotechnology is in thedevelopment of new strains of papaya. Agricultural biotechnology has been used tocreate virus-resistant papayas, thus saving the Hawaiian papaya industry. The papayaring spot virus (PRSV) has affected papaya production for decades, and traditional

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breeding methods have proven unsuccessful in solving the problem. However, in1986, with the discovery of a parasite-derived resistance gene that interfered withthe reproductive cycle of the virus, a modified crop was developed. After years oftesting, two seed varieties, ‘UH Sunup’ and ‘UH Rainbow’, were released to theindustry. Field trials of transgenic ‘UH Rainbow’ and ‘UH Sunup’ were establishedin Puna, in October, 1995, and they make up 1600 of the 3200 acres of papayasgrown today (Gonsalves, 1998; Shee, 2000).

Asgrow Vegetable Seeds, a California-based company, introduced a disease-resistantgreen-stemmed squash hybrid in June, 2000 (Packer, 2000). Three plant viruses—cucumber mosaic virus, watermelon virus and zucchini yellow mosaic virus—destroyup to 80% of U.S. squash crops during the growing seasons. Growers have planted ayellow-stemmed variety that is very susceptible to virus attack. University studies haveshown that the green-stemmed variety is more suitable for the fresh-cut industry,because it does not oxidize as quickly as the yellow-stemmed variety. The first twohybrids introduced, Destiny III and Liberator III, were produced by inserting genesthat improved the plant’s defense against viruses, interfering with the virus’ ability toreproduce. According to the Asgrow developers, the nutritional quality, taste andtexture of the hybrids remain unchanged, and U.S. regulatory agencies (the USDA,FDA and EPA) have already approved the products for consumer use (Shee, 2000).

Seminis Vegetable Seeds, of Oxnard, California, is using agricultural biotech-nology techniques to improve weed, insect and disease control in squash, tomatoes,melons, lettuce, peas and sweet corn. Traits that extend shelf life, allowing growersto harvest later in the growing cycle, while still obtaining product that can withstandabuses in distribution, will add value for growers, distributors, retailers and consum-ers (Shee, 2000). Seminis Vegetable Seeds continues work on shelf life stability oftomatoes and melons and nutritional content of tomatoes and broccoli. The companyplans to introduce these new products in 2003–2005 after extensive safety evaluationand field testing and after fulfilling regulatory requirements for approval. Moregenetically enhanced crops will be on the market in the future, as scientists continueto improve the properties of existing produce.

Other novel produce items being developed using agricultural biotechnology arefruits without fertilization. Focus has been placed on cherry, raisin tomato, squash,eggplant, pepper, strawberry, melon and watermelon. The benefits of this are improvedtaste, because seedless fruits often have higher total soluble sugar (TSS) levels thanseeded fruit. Benefits also include reliable crop yield, because pollination is eliminatedas a factor in fruit development. In greenhouse operations, there would be a decreasein pollination-related expenses. Processing plants benefit, because there is a longerharvesting period enabling processing equipment to be used more efficiently. The seedremoval stage is eliminated, thus reducing expenses. Eggplant has been produced usingthe auxin gene (iaaM) under the control of Antirrhinmum majus ovule-specific promoter.

Nutritional and Biomedical Benefits

Agricultural biotechnology will be used to improve nutrition and quality of peppers,strawberries, raspberries, bananas, sweet potatoes and melons. A major application willbe in strawberries, with a higher crop yield and improved freshness, flavor and texture.

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Reduced allergens, a reduction in natural toxin levels in plants and peanuts with animproved protein balance reducing allergic reaction are some of the many benefitsthat are projected to be derived using agricultural biotechnology. Other potentialcrops that will provide health benefits include the following (IFIC, 2000a):

• tomatoes with a higher lycopene content (antioxidant)• fruits and vegetables fortified with or containing higher levels of vitamins,

such as C and E, to potentially protect against the risk of chronic diseasessuch as cancer and heart disease

• enhanced allicin production in garlic cloves to help lower cholesterollevels

• increased ellagic acid, a natural cancer-fighting agent, in strawberries• improved rice proteins, using genes transferred from pea plants• simple, fast methods for detection of pathogens, toxins and contaminants

Rice, a staple in many countries of the world, has been improved, from anutritional standpoint, by the introduction of genes from daffodil and a bacterialstrain. Swiss professor Ingo Potrykus developed a rice strain that is able to produceβ-carotene, the precursor to vitamin A. This ‘golden rice’, as it has been named, issaid to have the potential of saving millions of children worldwide from blindnessand other illnesses associated with vitamin A deficiency. Potrykus collaborated withPeter Beyer of the University of Freiburg in Germany to make golden rice. Beyer,an expert in the biochemistry of daffodils, was able to provide the genes for makingβ-carotene. The scientific challenge of this development was the transfer of a groupof genes that represented a key part of a biochemical pathway, and not just a singlegene, as in previous work with transgenic crops (Nash, 2000). The genes that givegolden rice its ability to make β-carotene in its endosperm come from daffodils and abacterium called Erwinia uredovora. These genes and various promoters are insertedinto plasmids of the bacterium Agrobacterium tumefaciens. The agrobacteria are usedto transfect rice embryos, transferring the gene coding for β-carotene. The transgenicrice plants must then be crossed with strains of rice that are grown locally and aresuited to a particular region’s climate and growing conditions (Nash, 2000).

Starting with the precursor geranylgeranyl diphosphate, enzymes added includedphytoene synthase, from daffodil. Phytoene is the first carotenoid precursor in thebiosynthetic pathway leading to the production of β-carotene. Phytoene desaturasefrom Erwinia and lycopene β-cyclase from daffodil react with the precursor toproduce β-carotene. Also, ferritin, an iron-rich bean storage protein and phytase, anenzyme that breaks down phytate making Fe available, were added to rice. A genefor a cysteine-rich metallothionine-like protein, for reabsorption of iron, has alsobeen engineered into rice (Potrykus et al., 1998; Ye et al., 2000).

Funding for this project was initiated by Gary Toenniessen, director of foodsecurity for the Rockefeller Foundation. He identified the lack of β-carotene inpolished rice grains as an appropriate target for geneticists, because traditional plantbreeding seemed unable to address the problem. Later, funding was also suppliedby the Swiss government and the European Union. Because the work was carriedout without industrial support, Potrykus hoped to distribute the technology freely to

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countries that needed it the most. There are still major hurdles to cross, however,like patents and proprietary rights of genes used, as well as criticism by publicinterest groups like the Rural Advancement Foundation International of Canada andothers. The affiliation of golden rice with a large commercial enterprise, AstraZeneca,which will market the product, is felt to be contrary to public trust, because the cropmay not have been tested thoroughly for adverse human effects (Nash, 2000).

Another recent advancement in agricultural technology is the development ofpotatoes with higher starch content. The bacterial gene coding for enzyme ADP-glucose pyrophosphorylase was inserted into the potato genome under the controlof the patatin promoter, resulting in 30–60% greater accumulation of starch. High-starch potatoes have less moisture and absorb less fat during frying. This product,if commercialized, would be very beneficial in lowering the calories in french fries,one of the most heavily consumed foods in the United States, thus helping to alleviatethe national challenge of obesity.

Transgenic Plants as Vaccine Production SystemsTraditionally, fermentation-based systems have been used for industrial productionof vaccines, but genetically modified plants that express foreign proteins offer aneconomical alternative. Large amounts of antigens could be produced using agricul-ture instead of complex cell culture-based expression systems (Weksler, 2000). Theconcept of genetically modified plants being used for vaccine production emergedin 1992 when an investigation was conducted to produce different classes of proteinsof pharmaceutical value in plants. Also, the announcement of the Children’s VaccineInitiative in 1993 documented the need for vaccine technology to combat infectiousdiseases. For a review of the progress made by several groups of scientists in vaccineresearch using genetically modified plants, the reader is referred to Mason and Arntzen(1995).

Diseases have been extremely challenging in developing countries, where anti-gens from enteric pathogens were early targets for plant-based expression studies.Convenience and the need to evaluate genetic constructs quickly, determined theinitial plant systems used in testing recombinant antigen production. Tobacco plantswere used in early studies, but high levels of toxic alkaloids in the leaves inhibitedanimal feeding studies. Many scientists used potato, because mice accepted rawpotato tubers instead of feed, and these could be generated in a relatively short timeafter transformation.

Genes coding for antigens from pathogenic viruses, bacteria or parasites thathave been characterized, and for which antibodies exist, can be manipulated in twoways. One method is transient expression using viral vectors. If epitopes within theantigen are identified, DNA fragments encoding these can be used to constructchimeric genes by fusion with coat protein genes from a plant virus, e.g., tobaccomosaic virus (TMV) or cowpea mosaic virus (CPMV). The recombinant virus, orin the case of TMV and CPMV, even the viral RNA made in vitro from the plasmidclone, is then used to infect established plants. Virus replication and systematicspread allow high-level transient expression of the chimeric coat protein in mostplant tissues. The viral particles expressing the foreign epitope on their surfaces arethen purified and used for immunogenicity studies (Mason and Arntzen, 1995).

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Biotechnology and the Fresh-cut Produce Industry 357

Alternatively, the entire structural gene may be inserted into a plant transforma-tion vector between 5′ and 3′ regulatory elements. This allows the transcription andaccumulation of the coding sequence in all or selected plant tissues. The vector isthen used for the Agrobacterium-mediated transformation of plant cells, or for stableintegration of the expression cassette by other means, with regeneration of transgenicplants. The resulting plants contain the expression cassette with stable integrationinto the nuclear chromosomal DNA and can be used either for extraction and partialpurification of the foreign antigen or for direct feeding of plant tissues for assessmentof immunogenicity (Figure 11.2) (Mason and Arntzen, 1995). Thus, eating fruit canthen induce antibodies just like a vaccination, rendering the person immune to thedisease. Dr. Charles Arntzen at Cornell University is actively pursuing research toallow children to be immunized against debilitating diseases like hepatitis B, simplyby eating a modified banana.

Examples of stable genomic transformation using genes encoding foreign anti-gens include Streptococcus mutans spaA protein, hepatitis B surface antigen (HbsAg)and E. coli heat-labile enterotoxin B subunit and cholera-toxin B subunit. A patentapplication published under the International Patent Cooperation Treaty in 1990 wasthe first report of an edible vaccine. The surface protein (spaA) of Streptococcusmutans was expressed in tobacco plants to a level of about 0.02% of the total leafprotein. Oral immunogenicity of spaA produced in E. coli stimulated the productionof S-IgA in saliva. Examples of transient expression of candidate vaccines usingviral vectors include malarial epitope fusions with tobacco mosaic virus (TMV)capsid protein, Zona pellucida protein fusion with TMV capsid protein and cowpeamosaic virus capsid protein fusion (Mason and Arntzen, 1995).

Because vaccine antigens can be produced in plants in their native form, greatpossibilities exist in using this technology for food-based, “edible vaccines” thatwould be more economical for delivery in developing countries. Some concerns andchallenges that must be overcome before commercialization of this process includemaximizing the expression of antigenic proteins, stabilizing the foreign proteinduring postharvest storage in plant tissues and enhancing the oral immunogenicityof some antigens. Also, the possibility of allergy and immune tolerance of orallyapplied antigens must be addressed. A delivery system is needed to deliver the ediblevaccine as a medicinal product at the correct dosage and not as a routine food source.Also, a thorough study of the immunogenic dose response and antigen levels invarious foods is needed, so initially, animals may be the more likely target for edible-vaccine technology than humans (Arntzen, 1997a,b).

ENVIRONMENTAL BENEFITS

Agricultural biotechnology may be used to improve the environment, for example,in the development of plants that produce biodegradable plastics or plants that areable to absorb harmful substances from the soil. Phytoremediation of contaminatedsoils could be tackled by several methods: pollutant stabilization, containment anddecontamination. In pollutant stabilization, vegetative cover and soil conditions aremanipulated to reduce the environmental hazard, while decontamination occurswhen plants and their associated microflora are used to eliminate the contaminant

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358 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

from the soil. The reader is referred to Cunningham et al. (1995) for a review ofthis topic. Figure 11.3 summarizes the processes involved in phytoextraction of con-taminants from soil, removal of organic material and the phytostabilization of soilcontaminants.

FIGURE 11.2 Strategies for the production of candidate vaccine antigens in plant tissues.(Adapted from Mason and Arntzen, 1995.)

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Biotechnology and the Fresh-cut Produce Industry 359

Green-plant-based remediation has been used for years in the form of constructedwetlands, floating-plant systems in wastewater treatment and reed beds. Becauseplants are living organisms that require oxygen, water and nutrients in order to grow,certain limitations exist in plant tolerance to the toxin that must be removed, as wellas soil conditions of pH, salinity and texture. Phytoremediation is typically slower

FIGURE 11.3 Processes involved in the phytoextraction of contaminants from soils. (Adaptedfrom Cunningham et al., 1995.)

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than chemical or physical processes used to remove toxins. However, large areas ofimmobile contaminants may be amenable to phytoremediation that is far more costeffective than landfilling or thermal treatment.

Sebertia accuminata is a rare plant that hyperaccumulates toxic metals. Thisplant grows on outcroppings of metalliferrous soils, having a sap that is 25% nickelby dry weight. Also, Thlaspi caerulescens, a member of the Brassica family, canaccumulate up to 4% zinc in its tissue without apparent damage. Limitations in theuse of hyperaccumulators for remediation are that these plants usually accumulateone specific element, they grow slowly and have a small biomass, and agronomiccharacteristics of these plants are not yet well established. Effective genetic engi-neering of metal transport and tolerance plants requires a good understanding of themechanisms involved. However, metal tolerance has been obtained by introducingmetallothioneins and by the introduction of a semisynthetic gene encoding MerA(bacterial mercuric ion reductase). Genetically modified Arabidopsis can reduce thetoxic mercuric ion to mercury (Hg), which is then volatilized, with tolerance levelsup to 100 µM. Alterations in phytochelatins and metal-binding peptides also offerpossible tolerance mechanisms. However, mechanisms to increase the translocationof metals from the root to the shoot are needed. Phytoextraction could be quiteprofitable if metals of economic value, like nickel or copper, are targeted. In caseswhere volatile forms of inorganic contaminants (e.g., selenium as dimethyl selinide)are produced, this would eliminate the costs of harvesting and processing, makingthe process more economical (Cunningham et al., 1995).

Phytodegredation may only take place if organic contaminants are biologicallyavailable for absorption or uptake by plants, followed by metabolism of plant orplant-associated microbial systems. Investigations of plant-associated microbial sys-tems have shown bioremediation through the plant rhizosphere. The rhizosphereaccelerates rates of degradation for many pesticides, as well as trichloroethylene andpetroleum hydrocarbons. Research has been conducted to accelerate the degradationprocess of the plant-microbe interface. However, more work is needed to increasethe rate of metal phytoextraction and to develop new plant and soil managementpractices. In phytostabilization, the role of the plants is to increase the sequestrationof the contaminant by altering water flux through the soil, preventing wind and rainerosion and incorporating residual-free contaminants into roots. Future benefits ofagricultural biotechnology in phytoremediation will require the combination of tra-ditional disciplines in waste management with the tools of biotechnology.

OTHER BENEFICIAL CROPS — PLANTS AS BIOREACTORS

Genetically modified plants provide an economical alternative to using microbialsystems for the production of biomolecules. Synthetic saccharides and fatty acidsof nonplant origin may be synthesized in plants. However, plants may also bemanipulated to overproduce plant metabolites. One benefit to the overproduction ofheterologous proteins in plants is that expensive purification could be avoided if theplant material is used in human food or animal feed, allowing oral or topicalingestion. Table 11.7 depicts a summary of the use of plants as bioreactors for theproduction of lipids, carbohydrates and proteins (Goddijn and Pen, 1995).

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TABLE 11.7The Use of Plants as Bioreactors for the Production of Lipids, Carbohydrates and Proteins

Compound Origin of Gene(s) Application Plant Species Used

LipidsMedium-chain fatty acids

California bay tree Food detergent, industrial

Oilseed rape

Monounsaturated fatty acids

Rat Food Tobacco

Polyhydroxybutyric acid

Alcaligenes eutrophus Biodegradable plastics

Arabidopsis, oilseed rape, soybean

Saturated fatty acids Brassica rapa Food, confectioneries Oilseed rape

CarbohydratesAmylose-free starch Solanum tuberosum Food, industrial PotatoFructans Klebsiella

pneumoniaeFood, pharmaceutical Potato

Increased amount of starch

Bacillus subtilis Industrial, food Tobacco, potato

Trehalose Escherichia coli Food, industrial PotatoE. coli Food stabilizer Tobacco

Pharmaceutical polypeptides

Alpha-trichosantin Chinese medicinal plant

Inhibition of HIV replication

Nicotiana benthamiana

Angiotensin-I-converting enzyme inhibitor

Milk Antihypersensitive effect

Tobacco, tomato

Antibodies Mouse VariousAntigens Bacteria, viruses Orally administered

vaccinesMainly tobacco

Tobacco, tomato, potato, lettuce

Antigens Pathogens Subunit vaccinesEnkephalin Human Opiate activity TobaccoEpidermal growth factor

Human Proliferation of specific cells

Oilseed rape, Arabidopsis

Erythropoietin Human Regulation of erythrocyte levels

Tobacco

Growth hormone Trout Growth stimulation TobaccoHirudin Synthetic Thrombin inhibitorHuman serum albumin

Human Plasma expander Tobacco, Arabidopsis

Interferon Human Antiviral Oilseed rapeTobacco, potatoTurnip

(continued)

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Experiments with the Agrobacterium rhizogenes (hairy roots) have led to theproduction of bioactive compounds. They express root-specific pathways and haveshown stable production of alkaloids, polyacetylene, sesquiterpenes, naphthoqui-nones and other natural products. Hairy roots have been adapted for the overproductionof secondary metabolites and biotransformation of chemicals (McGloughlin, 2000).A short-day flowering plant was used to make the Ma-1 gene that would offer thebenefits to biomass and forage crops in which flowering is undesirable. Agriculturalbiotechnology may be used to create new plant genotypes with attributes that can beturned on and off at various stages in the growth season. Phytofluors of differentcolors will be used to “tag” proteins, enabling scientists to study interactions betweenmolecules within the cells (Clark Lagarias, University of California, Davis), but fewtools are available at present for this research.

Applied Phytologics, Inc., Sacramento, California, has prepared a malting grainmodel using rice. The rice is transformed with the desired protein, controlled by an α-amylase promoter in the aleurone layer. This crop is grown in the field, and grain isharvested at maturity. Seeds are imbibed to induce expression of the α-amylase promoterand production of the desired protein. Proteins are then extracted from germinatingseeds and purified (McGloughlin, 2000). This novel method of protein expression hasbeen implemented by Applied Phytologics, Inc., for a wide range of biotechnologyproducts, including blood proteins, bioactive therapeutic proteins, price-sensitive indus-trial enzymes, animal health products and enzyme-based bioremediation.

Environmental stresses like drought, salt loading, freezing and elevated temper-atures affect plant productivity. A gene that was transformed into wild-type Arabidopsishas been shown to confer to the seeds the ability to complete germination at coldertemperatures. The choline esterase gene from a soil bacterium was used to transform

TABLE 11.7The Use of Plants as Bioreactors for the Production of Lipids, Carbohydrates and Proteins (Continued)

Compound Origin of Gene(s) Application Plant Species Used

Industrial enzymesAlpha-amylase Bacillus licheniformis Liquefaction of starch Tobacco, alfalfa(1-3,1-4)-β-Glucanase Trichoderma reesei,

hybrid of two Bacillus species

Brewing Barley cells

Manganese-dependent lignin peroxidase

Phanerochaete chrysosporium

Bleaching and pulping of paper

Animal feed

Alfalfa

Phytase Aspergillus niger Animal feed, paper and pulp, baking

Tobacco

Xylanase Clostridium thermocellum

Cryptococcus albidus

Tobacco

Source: Adapted from Goddijn and Pen (1995).

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Biotechnology and the Fresh-cut Produce Industry 363

Arabidopsis (McGloughlin, 2000). Choline oxidase catalyzes the conversion ofcholine to betaine, a potent protectant molecule in bacteria, plants and animals, thusenhancing stress tolerance. Glycine betaine insulates plant cells against the ravagesof salt by preserving the osmotic balance, by stabilizing the structure of proteinsand by protecting the photosynthetic apparatus. Transformed plants were able togerminate at low temperatures and grow at elevated and low temperatures thatseverely limit wild-type growth rates. Plants engineered with the COR15a transcrip-tion factor may indeed have better cold tolerance. Betaine overproducing plants werealso able to grow under conditions of elevated salt. Choline oxidase (codA) from asoil bacterium tolerated saline and cold conditions. A salt tolerance gene frommangroves (Avicennia marina) has been identified, cloned and transferred to otherplants that were found to be tolerant to high salt concentrations; and a gene fromE. coli was also used to generate salt-tolerant transgenic maize (Liu et al., 1999; ThirdWorld Academy of Sciences, 2000).

Plants have been used to synthesize biodegradable plastics. Genetically modifiedArabidopsis expressed the polyhydroxybutyrate (PHB) pathway in the cytoplasm.Plants are, in general, suitable for the production of industrial enzymes. However, itis important that the protein of plant origin is more economical to produce than theproduct of microbial fermentation. Agricultural biotechnology will increase yields ofrice crops with the properties of improved nitrogen assimilation, increased sucrosehydrolysis, starch biosynthesis, increased O2 availability and modified photosynthesis.Success in the use of plants as bioreactors will rely on the availability of structuraland biosynthetic genes for specific biosynthetic pathways, improvement of productionlevels and increased knowledge of downstream processing (Goddijn and Pen, 1995).

FOOD SAFETY CONCERNS

HUMAN HEALTH AND ENVIRONMENTAL RISKS

Recombinant DNA technology is no longer obscure, because there are many practicalapplications and even high school kits for gene splicing experiments. Two majornonbiochemical realities or constraints that govern both the rate and the amount ofpenetration the products of biotechnology will have in the food industry are economicsand regulatory approval. New components will have to be more cost effective than theingredients they are replacing, and the reformulated and totally new products will haveto pass the tests of the consumer marketplace. Food safety concerns with transgeniccrops include the introduction of novel proteins that are allergenic, and geneticexchange of traits like insect and herbicide resistance from transgenic crops to weedsand other wild-type species. Unintended effects of genetic modification may be man-ifested as the expression of unknown or unexpected toxic or antinutrient factors orincreased production of known toxic constituents (Royal Society, 1999). Other impor-tant issues in applying genetic engineering to food include nutritional equivalency ofgenetically altered foods compared to their traditional counterparts and sensory accept-ability of genetically altered foods compared to their traditional counterparts.

Food allergens are caused by immunological responses to substances in foods,usually natural proteins in commonly allergenic foods like peanuts, milk and seafood.

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Symptoms of allergic reactions can be mild cutaneous or gastrointestinal symptomsor life-threatening anaphylactic shock. Because new proteins are formed when genesare introduced in GMOs, potential allergenicity of the new food should be assessed(IFT Expert Committee, 2000a,b). Release of GMOs into the environment allowsthe possibility of plants breeding with related species in the wild. This has beendocumented in canola fields of Saskatchewan, Canada. Canola shipped to Francefrom Canada was banned early in the year 2000 because it contained greater than1% GM grain. It was then discovered that the shipment was contaminated by pollenfrom a genetically modified crop that was grown more than 100 meters away. Anotherexample of contamination of “GMO-free” product occurred in a shipment of wheatthat was transported with genetically modified product. The wheat flour was usedto prepare breading for turkey cutlets, and when tested, the breading was positivefor genetic modification (Stram et al., 2000).

Consumer concern about the safety of genetically engineered organisms is notunfounded. In the late 1980s, the unregulated, genetically engineered nutritional sup-plement, L-tryptophan, produced by Showa Denko, Tokyo, caused some 27 deathsby eosinophilia-myalia syndrome (EMS) (Hoyle, 1992). The problem was tracedback to changes in a bacterial fermentation process involving a recombinant strainof Bacillus amyloliquefaciens. The actual changes that caused the problem are stillunknown, so the importance of safety assessment cannot be stressed enough.

It was felt by some organizations that the environmental issue is not addressedsufficiently in the FAO/WHO safety assessment of food from biotechnology. A pilotplant without containment in the former German Democratic Republic producedmodified Bacillus subtilis strains carrying α-amylase genes from Bacillus amy-loliquefaciens on a nontransmissible plasmid derived from Streptococcus. Biomassfrom the plant was dispersed in a sewage pond, exhaust air was not filtered andbacteria in the enzyme solution were not inactivated (Teuber, 1993). When the plantclosed down, it was found that 5–18% of Bacillus, Streptococcus, Micrococcus andStaphylococcus strains isolated from the environment carried erythromycin resis-tance. This occurred because the modified plasmid pSB20 was maintained in bacteriathat survived in sterile soil and river water. However, selection pressure may alsoexplain the persistence of resistance genes in bacterial populations (Teuber, 1993).

Hansen (2000) compared the development of agricultural biotechnology withthe creation of synthetic chemicals in the early 1900s. Synthetic organic chemistrymay be an extension of basic chemistry, yet the distribution of some novel chemicalslike PCBs, organochlorine pesticides and vinyl chloride into the environment yieldedunexpected results. These chemicals were found to be carcinogens, reproductivetoxins, endocrine disruptors or causative agents for other medical conditions, andthe Toxic Substances Control Act was passed by the EPA, requiring premarketscreening of synthetic chemicals. It is possible that the experience with syntheticorganic chemicals could be repeated with the introduction of novel geneticallyengineered food into the biosphere.

Key areas for concern include the scope of genetic material transferred, unnaturalrecombination, location of the genetic insertions and use of vectors designed to moveand express genes across species barriers. Foreign promoters and foreign marker genes,particularly genes coding for antibiotic resistance, are used. Also, genetic engineering

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Biotechnology and the Fresh-cut Produce Industry 365

allows the insertion of genes not only from widely different plant families, but alsofrom any organism or synthetic genes. This is felt to introduce new elements ofuncertainty. The genome is made up of genes that interact in complex regulatorypathways to maintain the organism, so the addition of new genetic material may endup destabilizing pathways. Likewise, the introduction of a new species into the envi-ronment may cause little or no change or have a catastrophic effect on the ecosystem.Unfortunately, these changes cannot be predicted reliably with the limited knowledgeof the biology of the introduced species (Hansen, 2000; Matzke and Matzke, 1995).

The effect of a gene on the whole organism is significantly governed by its location.Thus, the lack of control over location in genetic engineering is cause for unexpectedeffects. Conventional breeding involves the reshuffling of alleles of organisms that sharea recent evolutionary history, so the genes most likely function in the same location.One example of the unpredictable effects of genetic engineering and the location ofgene insertion is an experiment by Bergelson et al. (1998) with Arabidopsis thaliana,a plant from the mustard seed family. Several lines that exhibited the same trait ofherbicide tolerance were compared: one developed by conventional breeding methodsand two using genetic engineering. Researchers at the University of Chicago inducedherbicide (chlorsulphuron) tolerance (HT) into A. thaliana via mutation breeding andgenetic engineering. The surprising results were that the per-plant out-crossing ratewas 0.3% for mutant fathers (mutation breeding) and 5.98% for transgenic fathers,a 20-fold difference. They attributed this difference to the difference in location ofthe insertions of the gene, because the genetic construct was the same in all of theplants. The act of genetic engineering had transformed a species that was normallyan in-breeder to an out-crosser (Hansen, 2000).

Another example of an unexpected effect that may have been a result of thelocation of insertion is the experiment by Inose and Murata (1995), who insertednot a transgene, but multiple copies of a naturally occurring yeast gene. Scientistsfound that a threefold increase in the enzyme phosphofructokinase resulted in 40-foldto 200-fold increase in methylglyoxal (MG), a toxic substance that is known to bemutagenic, ending on the yeast line. The genetically engineered yeast cells had sig-nificantly altered metabolism, resulting in the accumulation of a toxic substance, andthe conclusion was that the position of the insertion was the cause.

Environmental concerns also include the introduction of herbicide-tolerancegenes to plants that then increases its weediness. A general agreement on whatdefines a weed includes (APHIS, 2000) the ability to germinate in many differentenvironments; discontinuous germination and great longevity of seed; rapid growththrough vegetative phase to flowering; continuous seed production for the length oftime of growth; self-compatibility but partially autogamous and apometic; ease incross-pollination, by either unspecialized visitors or wind pollination; rapid growthin favorable environments and seed production in a wide variety of environments;short- and long-distance dispersal capabilities; and vegetative production and easyregeneration from fragments. Vertical transfer of the new genes, i.e., out-crossingfrom the transformed cultivar to other domestic plants, occurs naturally in nature.

Scientists have also recently discovered lateral movement of genetic material,called horizontal gene flow. This is thought to be one way in which antibiotic resistanceor pathogenicity is passed around among bacteria. Cho et al. (1998) reported evidence

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that genes from a fungus had invaded 48 out of 335 genera of land plants surveyed.Jakowitsch et al. (1999) demonstrated that sequences from a previously unidentifiedtobacco pararetrovirus had repeatedly integrated itself into tobacco chromosomes.In the past, it was thought that plant viral sequences rarely, if at all, integrated intohost genomes. Thus, it is now recognized that genetic material can move laterallybetween species and exist for extended periods of time. Horizontal gene flow innature is only limited to a few microorganisms, and plants have evolved defensesagainst this, but genetic engineering can be compared as an enhanced version ofthis natural phenomenon. Numerous marker genes are used in genetic engineeringto facilitate the identification of transferred genes. Markers are typically antibioticresistant. One concern is that these antibiotic genes may move horizontally towidespread bacteria in nature, rendering them resistant to the antibiotic in question.

Hoffman et al. (1994) reported horizontal gene transfer from higher transgenicplants via the soil to a soil microorganism (Aspergillus niger). Scientists reportedcases of genetic transfer across taxa of eukaryotes. The main example suggesting atransfer over evolutionary time from unrelated taxa to higher plants was the case ofvertebrate hemoglobin and legume hemoglobin. The fate of the Monarch butterflyreceived a great deal of publicity, because their larvae died after feeding on milkweedpatches adjacent to cornfields that had been genetically engineered for insect resis-tance. Laboratory studies showed that when fed milkweed coated with pollen con-taining the insecticidal protein, the butterflies die. In vitro studies, however, did notaccount for the fact that the pollination cycle and the migration of Monarch butterfliesoccur at different times (Gorny, 2000). Also, pollen is not typically carried longdistances, because it is too dense to float well in air currents. Horizontal (nonsexual)transfer of transgenes from genetically engineered plants into other organisms is notyet well documented or proven.

Vectors have been designed to move and express genes across species andecological barriers. No special genetic elements are required to facilitate movementof genes in conventional breeding, because it involves the mixing of genetic materialfrom species that are sexually compatible. In genetic engineering, vectors are usuallyderived from efficient genetic parasites like viruses or genetic elements that canenter cell barriers. Plasmids move readily between barriers, and in plants, the tumor-inducing plasmid (the Ti-plasmid) of Agrobacterium is used for agricultural biotech-nology. The genes of Agrobacterium are not found in crops in nature except in thoseinfected plants with crown gall or hairy root disease. Vectors used in agriculturalbiotechnology usually contain a powerful promoter derived from the cauliflowermosaic virus (CaMV 35S promoter). This causes disease in plants of the mustardfamily. Virus promoters enhance the hyperexpression of transgenes to a higher mag-nitude than the organism’s own gene. The naturally occurring promoters in plantswould not effectively express desired traits of a transgene if one relied solely onthis mechanism. The CaMV promoter is so strong that one safety concern is that itwould not only turn “on” the transgenes, but that it would also affect other genesthousands of base pairs upstream and downstream of the insertion site and evenaffect the behavior of genes on other chromosomes. It is possible for a gene thatcodes for a toxin to be turned “on” (Hansen, 2000).

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SAFETY ASSESSMENT

Consumer groups feel that the FDA has a legal obligation to require mandatoryreviews of all genetically engineered foods before they go on the market and todevelop ways to screen for unexpected effects that could have health consequences.The predictable risks and potential risks of toxins, allergens, nutritional changes andantibiotic marker genes should also be addressed. Safety review should also bedeveloped through a process of notice and comment (Hansen, 2000). In general, thecriteria used in safety assessment in foods include the chemical composition, spec-ification of the product, nutritional/metabolic data and toxicological data. If a genet-ically altered organism is found to be significantly different from a traditional one,more comprehensive testing is required, i.e., toxicological tests and nutritional testsof the food product. Where potential allergenicity is suspected, comprehensiveanimal and laboratory tests should be conducted and, if necessary, limited humanvolunteer studies should be conducted.

Allergenicity of products of agricultural biotechnology is assessed following adecision tree process outlined by the International Food Biotechnology Council(IFBC) and the Allergy and Immunology Institute of the International Life SciencesInstitute (ILSI) (Metcalfe et al., 1996). Focus is placed on the source of the gene(s),sequence homology of the newly introduced protein(s) to known allergens, the immu-nochemical reactivity of newly introduced protein(s) with immunoglobulin E (IgE)antibodies from the blood serum of individuals with known allergies to the sourcefrom which the genetic material was obtained and physicochemical properties likedigestive stability of the new protein (IFT Expert Committee, 2000b). Other assess-ment criteria that have been suggested by the FAO/WHO (1991, 2000) include theevaluation of the functional category for the protein introduced and the level ofexpression of the newly introduced protein(s) in the edible portions of the improvedvariety. Allergy assessment is a key part of the overall food safety assessment ofgenetically modified food, and strategies for assessment should be updated contin-uously as new technology and methods become available (FAO, 1996; IFT ExpertCommittee, 2000b).

The Animal and Plant Health Inspection Service (APHIS), a branch of the UnitedStates Department of Agriculture is responsible for the evaluation of geneticallyengineered plants. Guidelines have been prepared for submitting petitions to APHIS,and a petitioner is required to submit in detail the following information on new,genetically modified crops:

1. Rationale or development of the petitioned crop2. The biology of the petitioned crop3. Description of the transformation system4. Donor genes and regulatory sequences5. Genetic analysis and agronomic performance6. Environmental consequences of introduction of transformed cultivar7. Adverse consequences of new cultivar introduction 8. References

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Description of the biology of the nonmodified recipient organism should includetaxonomy, genetics, pollination, evidence of reported weediness and discussion ofsexual compatibility with wild and weedy free-living relatives in natural crosses orcrosses with human intervention. Applications should include whether the crop orsexually compatible species is listed in the relevant publications of the Weed Societyof America. The source of recipient (cultivar name or accession number) and theweed status of its sexually compatible relatives is also required (APHIS, 2000).Applications should also include the identification of the lines that are to be con-sidered in the petition and the cultivars from which they are derived. If there aremultiple lines, each line must be identified. In virus-resistant plants, applicationsshould also include a section for information on the nature of the virus that providedthe sequences encoding the resistance phenotype.

Because some plants have known toxicants that may affect nontarget organismsand beneficial insects, e.g., tomatin in tomatoes, cucurbitin in cucurbits or gossypolin cotton, the applicant should determine whether the introduction of new genes inthese plants altered the level of toxicants. If the plants produce no known toxicants,a reference should be provided to support the claim. The toxicological data on effectsof the plant on nontarget organisms and threat to endangered species is required ifthere is a notable difference between the transgenic and nontransgenic plant levelsof toxicants. During field testing, it should be determined whether adjacent nonsexuallycompatible plants developed weediness, in the case of the transfer of an herbicide-tolerant gene. When a single plant has more than one phenotype modification, thenonly one petition should be submitted. For a complete review of the APHIS require-ments in filing a petition for approval of a genetically modified plant, the reader isreferred to APHIS (2000).

Health Canada has primary responsibility for food safety, under the broad author-ity of the Food and Drugs Act. Health Canada reviews products for safety and setsdata requirements that will allow them to make safety assessments of products.Agriculture and Agri-Food Canada works closely with Health Canada to ensure thatfood risks are identified in order to prevent a threat to human health and safety(Agriculture and Agri-Food Canada, 1993). Criteria to assess food safety of novelfoods were revised in September, 1994, in publications of Health and Welfare Canada.Detailed guidelines are provided in two volumes to assist the researcher in safetyassessment and guidance in notification of novel food products (Health and WelfareCanada, 1994). New food products produced by genetic engineering were dealt withon a case-by-case basis to establish the nature of regulation required. Geneticallymodified microorganisms and their products when submitted for approval shouldcompare favorably with the unmodified organism, should not be pathogenic or pro-duce toxic substances, the DNA introduced should not have harmful sequences, thevector used should be characterized and potentially harmful markers (e.g., antibioticresistance) should be absent or inactivated (Health and Welfare Canada, 1994).

Health risks of biotechnology products are also assessed under the CanadianEnvironmental Protection Act (CEPA), which came into force in June, 1988. The goalof CEPA is to protect the environment and human health from “potentially toxic”substances. Products of biotechnology and genetically modified organisms are assessedand controlled by CEPA under a proactive program that prevents manufacture or

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entry into Canada until the federal government assesses potential effects on humanhealth and the environment. Scientists at Health Canada and Environment Canadaassess whether or not a product is “toxic,” if its use should be banned or controlled.Under CEPA, a product of biotechnology may be deemed “toxic” if it poses a riskto human health and the environment (i.e., wildlife and flora). Organisms involvedand any genetic modifications must be accurately identified. Biotechnology compa-nies are required to provide information on the organism used, its past history ofadverse effects on human health, if any, and antibiotic resistance profile. Firms mayalso be required to test GMOs for pathogenicity using procedures that are valid forthe specific organism. The federal government continues to monitor the health effectsof products of biotechnology long after they have been initially approved for use(Health Canada, 2000).

In terms of international safety assessment of GMOs, the World Health Organi-zation (WHO), since its inception in 1948, has promoted scientific research in foodsafety and the development of principles and guidelines to be used by its MemberStates. In 1991, a joint FAO/WHO Consultation resulted in the publication of specificrecommendations for assessing the safety of foods produced by biotechnology, includ-ing genetically modified organisms. The safety assessment was to be based on sound,scientific principles and data, and be flexible enough to accommodate scientificadvances. The organisms that contributed genetic material should be identified tax-onomically and genotypically. Vectors should be constructed to minimize transfer toother microbes and selectable markers genes should not encode resistance to clinicallyuseful antibiotics. Pathogenic organisms should not be introduced into food.

In May, 2000, the WHO introduced a series of consultations addressing the safetyof foods derived from biotechnology, co-sponsored by the Food and AgriculturalOrganization (FAO). The first joint consultation held in Geneva, Switzerland, May29–June 2, 2000, was followed by another joint consultation that focused on aller-genicity, from January 22–25, 2001, in Rome, Italy. The WHO joined with the ItalianEnvironmental Protection Agency to host a seminar on the potential health hazardsof the release of GMOs in the environment. Previous WHO consultations related tofoods derived from biotechnology include the 1996 meeting to provide practicalrecommendations for safety assessment, the 1995 meeting for guidance on the conceptof substantial equivalence, the 1993 meeting on health aspects of antibiotic resistancemarker genes and the 1990 consultation on safety assessment strategies.

The Codex Alimentarius Commission (CAC), a subgroup of WHO, is chargedwith establishing standards and guidelines to protect consumer health. In the twenty-third session of CAC held in June, 1999, the Medium-Term Plan for 1998 to 2002was adopted. This plan allows for the development of standards for foods derivedfrom biotechnology or traits introduced into foods by biotechnology, where justifiedscientifically. An ad hoc intergovernmental task force was established to implementthe Medium-Term Plan, and this group first met in Japan in March, 2000. Also, theCodex Committee on Food Labeling has discussed recommendations for the labelingof foods derived through biotechnology, and these will be included in the CodexGeneral Standards for Labeling of Prepackaged Foods. For a summary of the activ-ities of the FAO/WHO consultations and international policy making on foodsderived from biotechnology, the reader is referred to WHO (1990, 2000a,b).

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FAO supports a science-based evaluation system that would objectively deter-mine the benefits and risks of each individual GMO on a case-by-case basis priorto release. The possible effects of biodiversity, the environment and food safetyshould be evaluated, and the benefits of the product or process vs. the risks shouldbe assessed. FAO expects that the national regulatory authority in each region beconsulted when releasing GM foods. After release of GM foods, careful monitoringshould continue to ensure continued safety to human beings, animals and the envi-ronment (FAO/WHO, 1991; FAO, 1996, 2000). Another international group, theOrganization for Economic Cooperation and Development (OECD), was formed in1960 to promote policies to achieve the highest sustainable economic growth of itsmember nations and to promote trade on a multilateral basis. In 1992, the OECDpublished a report on Safety Considerations for Biotechnology that was intended forthose carrying out safety evaluations of new foods or food components derived bymeans of modern biotechnology. The report is based on comparison of GM foodwith traditional foods that are safe for consumption and the principles consideredin making evaluations. Also, a task force was created for the safety of novel foodsand feeds (OECD, 1993, 1998, 2000).

THE U.S. PERSPECTIVE

Genetically engineered foods were the number six concern in the United Statesaccording to a series of surveys conducted by the Food Marketing Institute from1995–1997. Topics ranked in order of “serious hazard” include bacterial contami-nation, general food safety, chemical pesticides, nutritional quality, artificial preser-vatives then genetically modified foods. Consumer telephone surveys were conductedby scientists at North Carolina State University in 1992 when bovine somatotrophin(BST) was a serious food safety concern (Hoban, 1994). Investigations were alsoconducted with the Food Marketing Institute, Canadian researchers and the Inter-national Food Information Council (IFIC) over the years in North America, Japanand throughout Europe to determine consumer attitudes about biotechnology (Hoban,1996a,b, 1997, 1998; Hoban and Katic, 1998). Support for biotechnology has waveredbetween 70% in 1992 to 72% in 1998 in the United States. Demographic differencesshow that men are more positive than women in their evaluation of biotechnology.

In a May, 2000, IFIC survey of 1000 randomly selected consumers, 79% saidthey had heard or read about biotechnology, while only 2% admitted to being wellinformed about the technology (IFIC, 2000a). Forty percent thought that there werefoods produced through biotechnology in the supermarket now, and 54% said theywould be likely to buy produce if it had been modified by biotechnology to tastebetter or fresher. A higher percentage (69%) said that they would purchase producegenetically modified to resist insect damage and require fewer pesticides, and 40%supported biotechnology to enhance plants that yielded reduced saturated fat. TheFDA policy to only label products of agricultural biotechnology if allergens wereintroduced or if the food’s nutritional content was altered was opposed by 28% ofthose surveyed. When informed that critics of the FDA policy felt that labeling ofGMOs should be mandatory, even if no harmful effects were introduced, consumerstended to support critics 43%. However, it was felt by 55% that simply labeling that

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products contained ingredients of biotechnology did not provide sufficient informa-tion to make an informed decision (Hoban, 1996b; IFIC, 2000a).

Agricultural biotechnology may gain greater acceptance with the 40% who arein opposition, if it were not veiled in secrecy. Consumers should be exposed to thebenefits and disadvantages of the technology in order to make informed decisions(Jungmeyer, 2000). A Food Marketing Institute survey of consumer confidence infood safety over the period of 1996–2000 has shown a gradual decrease from 84%with complete confidence to 74% confident in the year 2000. According to a surveyconducted by GAP Research for Philip Morris and the American Farm Bureau, 57%of consumers support the use of biotechnology to improve the taste of foods, 65%support its use to improve the nutritional value of foods, 69% support its use toincrease food production and 73% support biotech to reduce pesticide use (IFIC,2000a). Consumer surveys in North America show a high degree of acceptance offoods derived from agricultural biotechnology. Consumers also have high confidencein the government and regulatory agencies to assure the safety of food.

From an economical standpoint, many experts believe that farmers face an uncer-tain future in terms of profiting from biotechnology. In the year 2000, farmers werereported as receiving only $0.24 on every food dollar invested (Gorny, 2000). Annualproduce sales total $80 million representing over 300 commodities in the U.S., whilethe produce seed business is valued at $500 million a year (IFPA, 2000). Thus, thelarge investment for agricultural biotechnology research is difficult to justify for sucha small annual income. Increased farm revenue could be offset by higher costs ofseed, especially if farmers have to segregate genetically modified plants from thosederived from conventional methods. Retailers have the most influence on price, andincreased production yields may not be beneficial to farmers in areas where a gluthas already depressed produce prices. Most of the benefits so far have been to seedcompanies that developed new product lines. The consumer has not necessarilybenefited from lower prices, thus, informative decisions must be made on how toapply agricultural biotechnology to benefit the consumer (Harvey, 2000).

The major issues that determine acceptance of products of biotechnology arepublic acceptance, which drives market demand, and regulation. These topics andlabeling of foods derived from genetically modified plants continue to dominate andimpact commercial planting of transgenic crops and consumption of geneticallymodified foods. The FDA’s May 2000 policy that requires mandatory assessment ofproducts of agricultural biotechnology, a review of methods used in detecting theseproducts and the setting of labeling standards, make the whole process more transparent,and the industry hopes this will boost consumer confidence in biotechnology.

AN INTERNATIONAL PERSPECTIVE

A United Nations’ estimate of the world population by the year 2050 is 7.8 billionor as many as 12.5 billion people, as compared to about 5.9 billion in 1997. Unfortu-nately, the surface area of the earth will remain the same, and we are left with thechoices of securing more land for agriculture or increasing output on the land thatis used at present for farming. Agricultural biotechnology may hold the most promise

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to increasing crop yield without damaging environmentally sensitive areas or clear-ing more rain forests for farming.

In 1984, the Rockefeller Foundation, a private philanthropic organization withthe aim of increasing crop yield of small farmers in developing countries, introducedthe International Rice Biotechnology Program, focusing on Asia. Rice is the mostimportant crop in developing countries, accounting for 80% of all calories consumedin Asian countries (IFIC, 1999; NRC, 2000b). The Foundation hopes to increaserice yields by 20% in Asia by the year 2005. Similar work was introduced in Africain 1988, where the problems of soil-nutrient depletion and yield losses caused bypests and diseases take priority. According to Gary Toenniessen, director of agricul-tural sciences for the Rockefeller Foundation, “The tools of biotechnology shouldbe developed for all major food crops, including those primarily grown in developingcountries and on marginal lands” (IFIC, 1999).

Another organization, the International Laboratory for Tropical AgriculturalBiotechnology (ILTAB), is developing transformation methods for application inviral disease control in tropical plants like rice, cassava and tomato. The first transferof a resistance gene from a wild rice species to a susceptible cultivated rice varietywas reported in 1995. The resistance gene for the bacterium Xanthomonas oryzae,which causes disease in rice crop, was transferred to several useful rice varietiesthat are cultivated on more than 24 million hectares worldwide. The hope of ILTABis that this research will assist farmers in developing countries to increase rice yieldsthrough the development of disease-resistant strains (IFIC, 1999).

Agricultural biotechnology research is being established and strengthened at thenational level and within international agricultural research centers. Several devel-oping nations have an emerging private-sector crop-biotechnology industry thatproduces hybrid seeds and micropropagated plants for commercial farmers. Thesemolecular biology research facilities are limited, however, and collaborate heavilywith the public sector and international partners. Table 11.8 summarizes internationalorganizations that transfer plant biotechnologies to developing countries. The Con-sultative Group on International Agricultural Research (CGIAR), with its Secretariatat the World Bank, helps to coordinate the efforts of several organizations to conductstrategic and applied research, facilitate technology transfer as well as deliveradvanced technology to farmers in the form of improved seed (CGIAR, 1999; Cook,1999; Toenniessen, 1995).

The CGIAR network has only had marginal success because of numerous chal-lenges. One challenge is the ability to produce improved varieties for highly variableland areas with limited agronomic potential, for example, in parts of Africa, LatinAmerica and Asia. On-farm research is needed to develop sustainable cropping systemsthat allow performance under local conditions. Also, in densely populated areas wherehigh yield varieties are widely used, there is little land left for expansion. Developmentof yield-enhancing and resource-conserving technologies will help to solve thesechallenges. Seed multiplication is relatively straightforward for cereals, but vegeta-tively propagated crops such as cassava, potatoes, yams, plantains and bananas requiremore technical biotechnology methods for improvement (Gould, 1999).

Traditionally, free exchange of materials and information has assisted in inter-national agricultural research. However, applied crop biotechnology research in

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TABLE 11.8International Organizations Facilitating the Transfer of Plant Biotechnology to Developing Countries

International Organization DescriptionFood and Agriculture Organization (FAO) of the United Nations, Rome, Italy

Conducts research and facilitates transfer of plant biotechnology that can benefit developing countries through its Plant Production and Protection Division in Rome, its regional offices and the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture in Vienna

International Atomic Energy Agency (IAEA), Vienna, Austria

Conducts collaborative research national agencies and provides training in mutation breeding and other plant biotechnology through the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture

International Laboratory for Tropical Agricultural Biotechnology (ILTAB), La Jolla, CA, United States

An advanced-research laboratory, developed through a collaboration between the Scripps Research Institute and the French Technical Assistance organization ORSTOM, which conducts research and offers training on development of disease-resistant tropical plants through genetic engineering

International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy and New Delhi, India

Originally established by the United Nations Industrial Development Organization and now an independent research and training organization with crop biotechnology programs in New Delhi and information dissemination provided through Trieste

Center for the Application of Molecular Biology to International Agriculture (CAMBIA), Canberra, Australia

A research and technology transfer orgnization specializing in the production and dissemination of inexpensive biotechnology tools that can be employed in developing countries

International Service for the Acquisition of Agribiotech Applications (ISAAA), Ithaca, NY, United States

An international organization committed to the acquisition and transfer of proprietary agricultural biotechnologies from the industrial countries for the benefit of the developing world

Intermediary Biotechnology Service (IBS), The Hague, the Netherlands

A unit of the International Service for National Agricultural Research that provides national agricultural research agencies with information, advice and assistance to help strengthen their biotechnology capacities

Biotechnology Advisory Commission (BAC), Stockholm, Sweden

A unit of the Stockholm Environment Institute that provides biosafety advice and helps developing countries assess the possible environmental, health and socioeconomic impacts of proposed biotechnology introductions

Technical Center for Agricultural and Rural Development (CTA), Wageningen, the Netherlands

A unit of the European Union that collects, disseminates and facilitates exchange of information on research innovations including plant biotechnologies for the benefit of Asian, Caribbean and Pacific states

International Institute for Co-operation in Agriculture (IICA), San José, Costa Rica

Assists countries in Latin America and the Caribbean with policy issues related to biotechnology including the formulation and harmonization of biosafety procedures

(continued)

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industrial countries has increasingly become the function of “for-profit” organiza-tions. There is a significant increase in research that is protected under many formsof intellectual property rights, including patents, plant breeders’ rights and tradesecrets. Now, even public sector plant scientists are being encouraged to seek intel-lectual property rights for their inventions and to license technology to the corporatesector. Even if information is shared, results needed for further distribution andcommercialization are retarded by one or more material transfer agreements. TheInternational Service for the Acquisition of Agri-biotech Applications (ISAAA) wasformed to serve as an agency to facilitate transfers of proprietary agricultural bio-technologies from industrial countries to the developing world (Toenniessen, 1995).

Developing countries may be faced with the dilemma of a second generation ofdependency on industrialized nations through the appropriation of germ plasm bythe latter and through socioeconomic dislocation resulting from substitution ofbiosynthetic products for natural ones. But, as former U.S. president Jimmy Carterstated: “Responsible biotechnology is not the enemy; starvation is. Without adequatefood supplies at affordable prices, we cannot expect world health or peace” (IFIC,1999). In order to meet the challenge of feeding the ever-growing populations indeveloping countries, it will be necessary to deliver low-cost, high-value seeds to poorfarmers and to ensure that crop germplasm can continue to be distributed and sharedamong the developing countries without restrictions. The sociopolitical and socio-economic obstacles limiting food distribution may be the real barriers to fooddistribution to the hungry (Gorny, 2000).

The United States is the leader in the growing of crops derived from agriculturalbiotechnology—the most predominant crops being GM corn, soy and cottonseed.During the year 2000, there was a modest reversal to traditional crops with a 9–24%decrease in biotech crops from the year 1999, especially in corn (Barach, 2000).Transgenic corn production went from 33% of total corn crop to 25%. This decreasehas been attributed to adjustments made to accommodate sales to Europe and countries

TABLE 11.8International Organizations Facilitating the Transfer of Plant Biotechnology to Developing Countries (Continued)

International Organization DescriptionBiotechnology Research Center, Chinese Academy of Agricultural Sciences, Beijing, China

Carries out research in crop breeding using tools of biotechnology; use marker-aided selection

Biotechnology Center, Indian Agricultural Research Institute, New Delhi, India

National Center for Research on Genetic Resources and Biotechnology, EMBRAPA, Brasilia, Brazil

Source: Toenniessen (1995), with permission.

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that have a non-GMO policy, as well as a reduced demand because of less insectpressure. Numerous processed and formulated foods contain GM soy, corn andcottonseed oil in products like salad dressings. Soy ingredients may be found in upto 60% of all formulated foods in the U.S. today, with consumers estimated to haveingested GM foods since 1996. The European market is very important to the UnitedStates, estimating $4 billion in sales of value-added food products in 1999. Therecent policy of non-GM foods would make export of food products to this regionextremely difficult. Most U.S. food processors do not formulate products exclusivelyfor the European marketplace. Recent restrictions in labeling and difficulties inreformulation or ingredient segregation have caused some U.S. processors to ceaseexports to the European Union (EU). It has proven easier for some companies to sellto other markets than to attempt to comply with EU labeling regulations.

Bulk items like soy that are processed into ingredients are handled without intentor opportunity for segregation of GM crops from identity preserved (IP) crops. Onlysmall, niche markets of specialty crops would have the service available to segregateIP crops. However, isolated grower and handling and certification and testing incurcosts that must be recovered in the finished product. The EU and Japan, as well asthe organic market, may be niche markets for IP crops. This mode of handling wouldnot be practical for commodity items. Only labeling may satisfy the consumer’s“right to know.”

There are social and cultural reasons for the greater opposition in Europe toagricultural biotechnology. Consumers are more skeptical and concerned, so newproducts face significant challenges to achieve a level of consumer confidence.Northern European Union nations and Spain view GMOs with less scorn than Italyand France, but the presence of Greenpeace has caused governments to be cautious.There is a general lack of confidence in government food authorities in the EU. Thiswas probably fueled by the highly publicized food safety crisis of mad cow disease(bovine spongi form encephalopathy or BSE) in the United Kingdom and dioxin-contaminated feed in Belgium. Also, there are few scientific and academic voicessupporting the safety of agricultural biotechnology (Anonymous, 1998). Anti-GMlobbyists have been more successful at communicating the risks of GM foods thanbiotechnology companies promoting the benefits. Consumer activist groups likeGreenpeace have gained a great deal of support in their activities against geneticallymodified food. Political action has raised awareness of GM food with some EUnations restricting the use and sale of GM corn and enforcing the labeling of GMfoods. Retailers have also influenced consumers by eliminating GM product whenpossible.

Reaction from consumers and industry across Europe is not uniform, however.Northern European nations tended to be more aware of the environment and focusedon consumers, thus tending to create some resistance to biotechnology, while south-ern Europe has shown an indifference. A survey conducted in Europe, Canada, theUnited States and Japan in 1995 showed that above 50% of consumers were willingto buy GM insect-protected produce. Two countries where acceptance was low, wereAustria (22%) and Germany (30%). Austrian consumers were some of the mostnegative toward agricultural biotechnology. But, Austria has a large number oforganic farmers and low government support for biotechnology, hence, the negative

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reaction is understandable (Hoban, 1997). Consumers in these countries were thelowest in their willingness to buy products of biotechnology; however, they had arelatively high awareness of the technology, because those interviewed respondedas having spoken to someone about GMOs. When consumers were asked to evaluateapplications of biotechnology in food, animal health and human healthcare, 85% ofpeople around the world found applications in healthcare acceptable.

Media analysis in countries that showed the most resistance to GMOs revealedthat the opponents to biotechnology were able to voice their opinions relativelyunchallenged by biotechnology companies. More positive media coverage only cameafterwards. Consumers seemed to need a basic understanding of how food wasproduced. When asked if ordinary tomatoes did not contain genes while geneticallymodified ones did, there was a great deal of uncertainty. Fifty percent of Americansdid not know, while there was considerable variation in knowledge among Europeancountries. Some of the perceived fears of GMOs became evident when consumerswere asked if consuming GMOs would change a person’s genes. There was a betterunderstanding in the Netherlands, Canada and the United States, while 40% ofrespondents from Austria thought this was true (Hoban, 1997).

Also, there were significant differences in the ability of various groups to educateconsumers. U.S. consumers trusted organizations like the American Medical Associ-ation, the National Institutes of Health, the Food and Drug Administration and universityscientists to supply trustworthy information. The media, biotechnology companies,packaged food manufacturers, chefs, and activist groups and retailers tended to havelower credibility. However, in Europe, the sources of consumer education were theopposite. European consumers trusted environmental and consumer groups more thanthe government and industry to supply trustworthy information. Ironically, these edu-cators have been the strongest opponents of agricultural biotechnology.

There is a major philosophical disagreement as to what agriculture should be:natural selection vs. genetic manipulation using new technology (Nuffield Councilon Bioethics, 1999). China is moving ahead with research in agricultural biotech-nology, with more than 1 million farmers planting genetically modified crops fromabout 200 seed varieties in the year 2000. The European Union, however, has addedonly 10 million hectares in Eastern Europe to its subsidy-dependent agriculturalsystem, refusing to accept agricultural biotechnology and putting restrictions on farmtrade (Karst, 2000). One concern in developed nations is that the proponents oforganic growth may impose the belief that this is the only acceptable method ofbreeding new plant species, thereby preventing novel research using the methods ofbiotechnology that could be of benefit.

LEGAL CONSIDERATIONS AND LIMITATIONS

REGULATION OF GMOS

New technologies present new safety concerns, and the products of modern biotech-nology inevitably will be subjected to regulation by the federal government forthe purpose of assuring safety. A basic general principle of food safety and nutritionis that food should be safe, sound, wholesome and fit for human consumption.

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Consumers and scientists are concerned that genetically modified organisms orproducts might harbor unknown risks for human health and the environment.

Setting up regulating procedures was a vital first step in controlling “novel” foodproducts, which were defined as food that had not been used previously to anysignificant degree for human consumption. In the United States, the federal govern-ment approved rules and guidelines for regulating the biotechnology industry inJune, 1986. Regulation of safety of new products was divided among five federalagencies. The Food and Drug Administration (FDA) was responsible for geneticallyengineered organisms in foods and drugs. The USDA was responsible for engineeredorganisms used with crop plants and animals. The National Institutes of Health andthe Occupational Safety and Health Administration (OSHA) were responsible forengineered organisms that could affect public health and the workplace, respectively.The Environmental Protection Agency (EPA) was responsible for engineered organ-isms released into the environment for pest and pollution control and related activities.

The principal statute administered by the FDA was the Federal Food, Drug andCosmetic Act (FD&C Act) (1982). There was also the Federal Meat and PoultryProducts Inspection Acts. Genetically altered plants were subjected to the FederalPlant Pest Act and other laws depending on how they were constructed and theirintended use (Wasserman, 1988). FDA believed its existing requirements and pro-cedures with respect to food additives and generally recognized as safe (GRAS)food substances were sufficient. The FDA stated that it would apply the existingrequirements and procedures to the products of modern biotechnology on a “case-by-case” basis. Under the FD&C Act, the FDA’s principal power to regulate foodapplications of modern biotechnological methods was found in its premarket clear-ance authority. Such clearance powers were tied to legal classification of the foodingredient, namely, whether it was a food additive, a GRAS substance, a prior-sanctioned ingredient or simply a food. The FDA’s authority to require clearancebefore entry in the marketplace was confined to food additives only.

The FDA published its policy on genetically modified foods in 1992, establishingthat the FDA would treat as equivalent, food derived from plants modified by olderbreeding techniques and those derived from plants modified by genetic engineering.According to the policy, certain foods would be considered food additives under theFD&C Act. Under this policy, however, the FDA did not require premarket reviewif the food constituents of the new plant variety were the same or substantiallysimilar to those in other foods. The FDA required that the following concerns ofnovel genetically modified foods be addressed:

1. Does the food contain genes from known allergenic sources?2. Have novel toxins been introduced or have endogenous toxins increased? 3. Has the nutrient content changed?4. Does the food contain a substance that is new to the food supply?

Premarket approval was required when the characteristics of the new varieties posedfood safety questions from novel ingredients or toxicants. The policy also addressedthe introduction of allergens that were not present previously. In cases of seriousallergenicity risks, such foods would be banned from the food supply. All genetically

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modified foods introduced from 1992 to 1999 went though the premarket approvalprocess voluntarily.

Recent lobbying by antibiotechnology groups has forced a change in U.S. Foodand Drug Administration (FDA) policies. All new genetically modified foods mustface mandatory assessment, even when there is little or no scientific basis for a formalassessment. The Clinton administration announced in May, 2000, that food biotech-nology rules would be developed and implemented that would allow for moregovernment oversight of genetically modified crops. Also, labeling standards wouldbe set for foods marketed as “biotech-free.” It would become mandatory to providethe FDA information about new genetically engineered crops and notify the agencyat least four months before introducing the product on the market. The USDA, whichoversees the growing of genetically engineered crops, requires a buffer zone aroundcrops to make sure pollen drift does not occur.

The USDA will be responsible for building a certificate program for new scientifictests that detect products of agricultural biotechnology (Barach, 2000). Senator DickDurbin of Illinois in October, 2000, began promoting the regulation of food producedusing techniques of biotechnology. The proposition would allow the FDA to have theauthority to approve new foods and determine if they contained genetically modifiedelements. Labeling of genetically modified foods is now voluntary in the FDA’s policyand is only considered necessary if foods contain allergens or toxins. The proposedlegislation does not call for mandatory labeling, and the topic would be reviewed byCongress. In Canada, the Committee on Voluntary Labeling, an initiative by theCanadian Council of Grocery Distributors, drafted a document that included topicssuch as requirements for claims, claim templates, compliance measures and verifi-cation (Waterfield, 2000). For a thorough review of this topic, the reader is referredto the Institute of Food Technologists, Expert Report on Biotechnology and Foods(IFT Expert Committee, 2000c).

An important legal consideration is the evaluation of food for the presence ofgenetically modified organisms. At present, a number of European nations havedeclared that they will not accept products derived using agricultural biotechnologymethods. Testing is conducted to ensure a less than 0.1% GMO content (DNA and/orprotein content). There is no validated method or common international standard ofmeasure, and, in some cases, test results have proved to be inconsistent between labsin the United States and abroad (Roseboro, 2000). The most common method usedto detect GMOs is the polymerase chain reaction (PCR), which analyzes at the DNAlevel. This method is thought to be sensitive and able to quantify GM content at lowlevels. There are, however, no common protocols to address the many factors influ-encing PCR reliability, like sample preparation, DNA extraction, PCR amplificationof genetically modified sequences and electrophoretic analysis of PCR reaction prod-ucts to determine the presence and concentration of genetically modified DNA.Enzyme-linked immunosorbent assay (ELISA) is used to detect target proteins.

PCR is not easily adapted for rapid on-site testing at elevators or processingplants, because specific DNA sequence information is needed to prepare primers, andthis may be proprietary. Also, an initial investment of $20–30,000 for conventionalPCR, to $60–100,000 for real-time PCR is quite costly and requires highly skilledtechnicians for operation. ELISA tests analyze for a specific antibody reaction marking

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the presence of the expected protein. This method is less expensive and can be carriedout by nontechnical personnel, allowing testing at the point of sale. Several privatelabs have been established for GMO testing. One of the pioneers, Genetic ID, receivedaccreditation from the United Kingdom Accreditation Service (UKAS) for all GMOtesting methods. The European Union, one of the major markets requiring GMO testing,accepts UKAS accreditation. The USDA’s Grain Inspection Packers and StockyardAdministration (GIPSA) plans to accredit U.S. PCR laboratories for testing grains.This is a move to ensure that participants in the trade of grain feel more confidentabout their transactions. Also, collaborative studies between the American Associationof Cereal Chemists and the Association of Official Analytical Chemists will be con-ducted to evaluate PCR methods and laboratories’ ability to quantify the GM contentof grain samples and processed food (Roseboro, 2000).

The American Crop Protection Agency (ACPA) will support the U.S. Departmentof Agriculture’s initiatives to develop processes and procedures for the identificationof biotechnology-derived crops by providing reference materials and methods thatwill allow for more accurate testing. Also, plant biotechnology companies will assistthe USDA in efforts to validate tests for detection of biotech crops. In May, 2000,the USDA announced that it would seek public comments on proposed testingvalidation and accreditation standards. Plant material containing specific proteinsexpressed by transgenic genes and the DNA sequence for the novel trait havetypically been considered proprietary, so a consensus is needed from plant biotechcompanies to make standard reference material for transgenic crops accessible fortesting. ACPA member companies have agreed to assist in this process. For anupdated report on the methods of detection of GMO grain in commerce, testingcosts and verification of grain as “GMO free,” the reader is referred to ACPA (2000).

Several European bodies have published guidelines for the detection of geneti-cally engineered foods. The International Life Sciences Institute Europe (ILSI) helda workshop in Belgium in 1998 to review current knowledge of detection methodsfor GMOs. Also, the Joint Research Center in Ispra, Italy, validated a PCR screeningmethod for detection of DNA of GMOs that year. Members of the AnalyticalEnvironmental Immunochemical Consortium (AEIC) have also developed a guide-line document for the use of immunoassays to detect proteins of GM crops. Currentregulations are based on quantitative PCR for DNA detection or ELISA for proteindetection. Food-processing steps like heat, pH treatment or enzyme reactions mayaffect the quantities of DNA and protein in food.

A new brand of identity-preserved (IP) corn products was launched by Cargill’sIllinois Cereal Mills, with a system called InnovaSureTM for producing such products.InnovaSureTM is a system designed to maintain the integrity of specialty whole corn,yellow goods and masa through comprehensive procedures that start with the seedand continue through to the customer’s doorsteps (Giese, 2000). The system requiresworking with seed companies, growing all crop on contract, providing IP protocoltraining and continuous in-house testing.

A food safety crisis in the United States involving the detection of ‘StarLink’ cornin taco shells, has raised questions about the feasibility of segregating geneticallyengineered foods from those produced using traditional methods of breeding. TheFood and Drug Administration had approved the use of StarLink corn for animal

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feed but had not moved in a timely manner to approve its use for human consumption.This may have been the cause for the confusion. About 2600 growers produced thecorn, and officials blamed Aventis, the developers of the new strain, for allowing itto enter the food chain. The USDA was forced to “arrest” millions of bushels ofcorn until it could be channeled for nonfood use. There has been a long record ofsafety of biotech foods since about 40 new items have been approved and in use forthe last 10 years. But, the USDA has made efforts to remove StarLink corn fromcertain export markets and out of the organic food market and other niche marketsthat do not want products of agricultural biotechnology (Waterfield, 2000).

Several international organizations are also involved in the regulation of productsof agricultural biotechnology in their respective countries. Scientists at the NationalCenter for Research on Genetic Resources and Biotechnology (Empraba) in Brazilhave proposed a statistically significant method for evaluation of substantial equiv-alence between a genetically modified crop and its conventional analog or wild type.The method expands on that proposed by the U.S. Department of Agriculture andis thought to provide a more significant comparison (Belem, 2000). Substantialequivalence is required to ensure the health and well being of the consumer, ratio-nalize the cost of the investment in developing the new product through biotechnol-ogy and provide the basis for regulation of products of agricultural biotechnology.

LABELING OF GMOS — THE DEBATE

Labels are used to convey information on nutritional or health-related concerns ofthe contents of a package. The FDA requires labeling in two instances: if the foodcharacteristics significantly differ from what is normally expected or if safety issuesarise because of the new technology. The principal criteria considered are as follows:

1. The presence of novel allergens2. Introduction of novel toxins or the increase of endogenous toxins3. The change of levels of important nutrients4. Significant alterations in composition

If the product of genetic engineering is identical to the normal version of the samefood, then the label could be misleading.

Consumer groups advise that labels will allow the consumer the right to choosewhich product he wants. They feel that the FDA decision over the term “significantdifference” should be irrelevant. They say a small difference or a large differencein products of agricultural biotechnology should not alter a decision in labeling.Labeling should not be a deviation from previous FDA policy. The FDA requiresthe labeling of frozen peas and fresh peas so that consumers can make a choiceabout what to buy. Thus, food produced by or derived from a process of geneticengineering where foreign DNA has been introduced into an organism would alsobe labeled, allowing the consumer the opportunity of choice (Consumer Union’scomments to FDA on Docket No. 99N-4282) (Hansen, 2000).

Some consumers justify labeling because they believe that the insertion offoreign DNA into food is an adulteration or contamination of food with a chemical.

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It is found that consumer concern is often quieted when they are educated that allliving things contain DNA; DNA is an ingredient in almost all foods. Proponentsof labeling could argue that all foods containing chemicals carry a generic labelstating that the products contain chemicals. However, this would be meaninglessand would not provide information to make an informed choice (McHughen, 2000).

In October, 2000, ‘StarLink’ corn product was discovered in food exported to Japanwhere mandatory labeling and import notification for foods containing GMOs willsoon be a requirement. Media exposure over the commingling of StarLink corn withvarieties approved for food may well prompt the mandatory labeling of all geneticallymodified foods in the United States, and this labeling of genetically modified foodscould increase product costs 16–18%. The government would also be challenged todetermine how much labeling should be done, what should be put on the labels, andhow to enforce labeling. In an IFIC survey, it was found that consumers did not placemuch value on labeling and said that they should not have to pay more to keep foodsegregated or labeled. However, this cost would not be borne by food companies, butindeed passed on to the consumer. If educational information is not provided onlabels, they are thought to be useless, but labeling remains a focus point for activistgroups. A review of the debate on food labeling was published by the Institute ofFood Technologists (IFT), Expert Committee on Biotechnology (2000d).

International debate on the labeling of GMOs has continued for a decade. In1990, the announcement of a genetically modified baker’s yeast in Great Britaincaused a public outcry. The Food Advisory Committee (FAC) of the UK subsequentlydesignated four basic food categories as a primary screening mechanism to determinewhen specific food labeling might be required. The groupings were as follows(Teuber, 1993):

1. Nature-identical food products of genetically modified organisms2. Food from intraspecies genetically modified organisms3. Novel food products of genetically modified organisms4. Foods from transpecies genetically modified organisms

Several barriers to the trade of genetically modified crops now exist in Europe.The European Union safety assessment review process was put on hold since early1998. No new products have been approved, and this has impacted trade with theUnited States. In addition, the EU requires mandatory labeling of products containinggenetically modified organisms. This labeling has been viewed by some as a pro-tectionist move to steer consumers away from this technology, rather than to educate.Since the institution of mandatory labeling, retailers in the EU have removed productfrom the shelves or reformulated product to exclude genetically modified ingredients.This has left the EU consumer with a decrease in choices. The elimination of tradeopportunities for commodity corn and soy has raised questions about these technicalbarriers to trade with U.S. companies doing business in the EU (Barach, 2000).

Other countries are also moving toward mandatory labeling as seen in Table 11.9.Australia, Japan, Korea, Russia and others are all working on labeling regulations.Each country has its own labeling criteria and unique labeling language, thus com-pounding the complexity of the situation. There is also a lack of infrastructure for

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TABLE 11.9International Status Summary in June, 2000, on Labeling for Products Containing Genetically Modified Organisms (GMOs)

Country Labeling Status Special ProvisionsUnited States For safety, nutrition

or allergen reasons (only when not substantially equivalent)

FDA policy adopted 5/92 Labeling of characteristic (not of process or GM origin)

Canada Similar to U.S. Policy adopted 12/95 9/99 initiative to develop guidelines for voluntary labeling

Mexico Senate passes bill to mandate labeling in 4/00 (requires approval by the House and Presidential Signature)

Australia and New Zealand

Proposal to mandate labeling released 11/99

Health Ministers postpone “decision” on labeling

Proposing strict verification standards for “GMO-free” or “sourced from non-GMO”

European Union Mandatory labeling EU Directive 258/97, 1139/98

Effective 9/1/98 Implemented 4/9/00

Does not permit “may contain” (no action on “negative list,” or analytical standards)

Standing Committee amendments to 1139/98 to establish de minimis threshold

10/99—1% de minimis threshold for identity preserved non-GMO product

Japan Proposal to mandate labeling released 11/29/99

Implementation date 4/1/2001

Requires verifiable system for segregation (no final threshold established)

Provides labeling for 24 products/product categories

China No labeling requiredRussia Labeling proposal

released 12/1/99Implementation date 7/1/00

Provides for a “negative” list

Source: Reprinted from Barach (2000), Food Testing and Analysis, June/July, with permission of thepublisher ©2000. The Target Group.

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compliance and education of consumers at present. The Cartagena Biosafety Protocolwas an agreement among 130 nations passed early in the year 2000 to regulate thetransboundary movement of living genetically modified organisms. The ratification ofthe agreement by 50 countries will establish an international framework for countriesto use when making decisions on introducing GM crops to farmland. However, becausea “precautionary principle” has been adopted in the Biosafety Protocol agreement,some countries may decide to exclude GM crops without any scientific evidence ofharm (Barach, 2000).

The Cartagena Protocol may not take effect in individual countries for anothertwo years, but it does not override rights and obligations under other internationalagreements like that of the World Trade Organization (WTO). Several key areas inthe agreement that may be problematic to trade with the U.S. include the fact thatexporters must obtain advance permission from the importing country prior toshipping products of agricultural biotechnology that will be released into the envi-ronment, commodity shipments that contain living modified organisms (LMOs) mustbe labeled and international labeling initiatives may force the United States toinstitute labeling laws.

Some nations are interested in factors other than science on which to base foodregulation, for example, ethical, social and cultural issues. Many are focused on theconsumer “right-to-know” concept. The Codex Alimentarius, the agency of the UnitedNations that deals with food safety, quality and labeling standards and guidelines,has a subcommittee on food labeling, which is working on standards for labelingfoods from biotechnology. U.S. trade associations like the National Food ProcessorsAssociation (NFPA) believe that labeling should only be used if it refers to safety,nutritional value, health or composition of the food. They also support voluntarylabeling, provided the information is truthful and not misleading. Voluntary labelscould state “Biotech Free” or “Contains Biotech Ingredients.” The EU consumer“right-to-know” provision goes one step further in claiming that the consumer mustknow if the process of genetic engineering was used in developing the crop, andthus, any ingredient derivatives must be labeled.

The EU Novel Foods Regulation (358/97) is the route for mandatory labeling,with the requirement that recombinant DNA and/or detectable protein products frommodified DNA are present. This ruling has been changed by EC Regulation 1139/98to require labeling of foods containing GM soy or corn. The EC Regulation 1139/98was further amended to establish a 1% de minimus threshold for inadvertent con-tamination of non-GM food. Labeling of such foods and ingredients containingadditives and flavorings is required (Barach, 2000).

There are, however, flaws in the labeling approach, because no threshold has beenestablished for the point at which labeling becomes unnecessary for DNA or protein.Refined ingredients that are known not to contain DNA and/or protein, e.g., oils andsugar, should form a list of products that do not require labeling. Standards are neededfor “Biotech Free” claims, and testing methods for determining the presence of GMOsshould be standardized (Barach, 2000). The impact of the apparent deficiencies inlabeling rules and regulations will be compounded when other countries introducetheir regulations. One example is the Australian-New Zealand Food Authority’s(ANZFA) five-level labeling approach with choices including “GM free,” “not sourced

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from GMO,” “may contain GM ingredients,” “contains GM ingredients” and “genet-ically modified.” As detection methods advance, the industry may continue to requirelower detection levels, and the labeling may become more complicated.

CONCLUSIONS

Agricultural biotechnology brings together the diverse fields of molecular biology,practical agriculture, sustainable agriculture and chemical engineering. Scientistsare now able to manipulate living organisms with greater precision than ever before.New advances in biotechnology have improved food production, increasing yieldsand making production more cost effective. Agricultural biotechnology has assistedfarmers with the introduction of pest-resistant crops that eliminate the need to applypesticides, saving the environment from harmful chemicals as well as reducingproduction costs. This new technology has also introduced herbicide resistance incrops, so that herbicides can be applied without the fear of crop destruction. Thesetwo main benefits of agricultural biotechnology have been used widely in sustainableagricultural crops like corn, soybean, wheat and potatoes. Experiments that benefitthe fruit and vegetable industry are in the early stages of development. Viral diseaseresistance in papaya and delayed fruit ripening in tomatoes are early examples ofhow biotechnology can be used to provide consumer benefits. In the long run,genetically engineered foods may minimize seasonal and geographic variations.Consumer trends are dictating the direction in which research should go. The accentthese days is on a more healthy and nutrition-conscious lifestyle. This has encouragedthe development of natural food products with enhanced nutritional benefits like“golden rice” which produces β-carotene. Extension of shelf life for the fresh-cutproduce industry will also make demands on current technology. Longer shippingtimes are required to further markets than traditionally.

Genetic engineering in plants differs from conventional breeding in that con-ventional breeding relies on selection, using natural processes of sexual and asexualreproduction. Genetic engineering utilizes a process of insertion of genetic materialvia a method of direct gene introduction. However, genetic engineering relies on theinsertion of the gene in a random location, which may disrupt the natural functionsand complex interactions in a plant cell, exhibiting unexpected effects. The use ofviral promoters, genetic material from Agrobacterium and bacterial antibiotic markergenes also introduces new variables with a cause for concern, for example, the intro-duction of new proteins that cause human allergic reactions. Release of transgeniccrops into the environment concerns consumer advocate groups and organic farmers.Increased weediness may develop if herbicide-resistant genes are transferred to wild-type plants, and the transfer of antibiotic resistance to organisms in nature couldcause future problems.

No evidence has yet been presented to prove that consumption of foods derivedfrom biotechnology produce adverse human health consequences. Since the applica-tion of the technology is relatively new, only after several generations may we becomeaware of the health risks, if any. Most science experts agree that biotechnologyderived foods are safe provided that they are “substantially equivalent” to the originalproducts. The USDA and FDA have set guidelines for the safety assessment of foods

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derived from agricultural biotechnology in the United States. Various internationalorganizations like FAO, WHO, OECD and the Codex Alimentarius Commissionhave proposed guidelines for safety assessment and labeling of these novel foods.Genetically modified foods may be the most highly regulated and observed foodsof all time. Ironically, crops produced by conventional breeding could technicallybe considered genetically modified, because the genes of the final product aresignificantly altered compared to the parent plant. Yet, these crops have traditionallybeen assumed safe for consumption.

Consumer acceptance of products of agricultural biotechnology vary from coun-try to country. The United States at present has the highest acceptance of geneticallymodified foods. In the European Union, there is a greater distrust of this newtechnology, fueled by numerous presentations of the negative aspects of the tech-nology by consumer advocate groups. The media in Europe has played a large rolein distribution of information on the dangers of biotechnology. Because there hasnot been an equally active campaign on the benefits of biotechnology, a great dealof fear and distrust has evolved. Consumer education is thought to be the main keyto acceptance of biotechnology, however, the issue becomes one of ethics vs. science.Is it ethical to tamper with nature and rearrange genetic information in organismsfor the benefit of man? There is no winner in this argument.

The European Union was the first to restrict the imports of genetically modifiedfoods. The member nations have introduced mandatory labeling laws so that theconsumer has the right to choose which product to consume. Many feel that thelabeling laws are unjust, because they create a fear of genetically modified foodwithout proof that there is a food safety hazard. There is an increase cost to labeling,and labeling laws are not yet clear on wording or enforcement methods. Othercountries have followed the EU by restricting the entry of GM foods. This has createdproblems in trade with the United States, the largest producer of GM crops forsustainable agriculture. Fresh produce is at the head of the organics market, forexample, in the United Kingdom, it accounts for about 40% of the market. Therehas been little focus on fresh produce in the debate of genetically modified foods.Price, quality and appearance still dominate; however, the fresh produce industrywould have to place great emphasis on consumer and retailer education in order topromote the benefits of genetic modification when the need arises. The fresh produceindustry will eventually be pulled into the debate of genetically modified foods aswhole foods replace processed foods as an area of concern.

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Flavor and Aromaof Fresh-cut Fruitsand Vegetables

John C. Beaulieu and Elizabeth A. Baldwin

CONTENTS

Introduction and OverviewFresh-cut Physiology and Flavor and AromaFlavor PerceptionDoes Fresh-cut Quality Indicate Flavor Quality?

Flavor Compounds in Fresh-cut ProduceVolatile Precursors and Biogenesis

Esters Aromatic CompoundsAldehydes and the Lipoxygenase Pathway

Important Aromas and FlavorsSugars (Soluble Solids), Organic Acids and Titratable Acidity

Factors Affecting Fresh-cut FlavorMorphological ConsiderationsChilling Injury and Storage TemperaturesVarieties, Growing Region and SeasonRipeness at Cutting, Firmness and ProcessingChemical and Physical Treatments

Chlorination and WashesCalcium Salts and AntibrowningAntimicrobial, Edible Coating and Other Treatment Compounds

Controlled Atmosphere, Modified Atmosphere Packaging and FlavorFlavor Life vs. Shelf LifeConclusion and Future ResearchReferences

12

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INTRODUCTION AND OVERVIEW

Throughout the last half of the 20th century, numerous investigations set out toidentify volatile compounds in many horticultural crops. Massive amounts of volatiledata have been generated for most commodities. However, we only have a relativelycomplete understanding or knowledge of the essential flavor compounds for someof our most popular fruits and vegetables. Much less work has been performed toelucidate the aroma-forming mechanisms. In this chapter, we discuss the genesisand importance of flavor attributes, considering the effects of processing and storageon selected compounds in some fresh-cut market products. Also reviewed are flavorattributes in relation to sugars, organic acids and titratable acidity (TA) in certaincommodities where these compounds are essential contributors to flavor. There is cur-rently little analytical and sensory data available concerning aroma and flavorchanges for fresh-cut commodities. Therefore, we took the liberty to discuss numer-ous items in regard to unprocessed fruits and vegetables and their storage, andextrapolated to confer logical physiological consequences of processing plus storagein fresh-cuts. Various preharvest and postharvest factors that may affect fresh-cutflavor quality will be addressed, where data are available. Flavor and sensory infor-mation in the literature will be used in conjunction with recent fresh-cut dataemerging in the literature and our laboratories.

F

RESH

-

CUT

P

HYSIOLOGY

AND

F

LAVOR

AND

A

ROMA

Fresh-cut processing causes major tissue disruption as vacuolar, cytoplasmic andnucleic enzymes and substrates become mixed (Watada and Qi, 1999; Wiley, 1994;Watada et al., 1990). Processing increases wound-induced C

2

H

4

and respiration rates,surface area per unit volume and water activity (King and Bolin, 1989). Additionaldetails concerning the physiological effects resulting from processing can be foundin Chapter 5. Physiological changes may be accompanied by browning, flavor loss,rapid softening, shrinkage and a shorter storage life. Accelerated water loss andincreased water activity and carbon supply from freed soluble sugars enhance poten-tial microbial attack, especially in fruits. Therefore, flavor and texture changes/lossduring and after processing are especially of concern in fresh-cuts, yet little researchhas occurred in this area. This review subsequently outlines important pathways andenzymes believed to be critical concerning genesis of volatile flavor compounds orclasses of compounds. Thus, information regarding flavor genesis will be morereadily available to the fresh-cut industry.

F

LAVOR

P

ERCEPTION

Flavor is an important internal quality factor for fresh produce. Consumers oftenbuy the first time based on appearance, but repeat purchases are driven by internalquality factors such as flavor and texture. Flavor is comprised of taste and aromarelating mainly to sugars, acids and volatile components (DeRovira, 1996). Humanperception of flavor is exceedingly complex. Aroma compounds are detected byolfactory nerve endings in the nose (in parts per billion) (DeRovira, 1997). In contrast,taste is the detection of nonvolatile compounds by several types of receptors in the

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tongue (in parts per hundred). The brain processes all of this information to give anintegrated flavor experience. However, the brain may interpret changes in aroma aschanges in taste (O’Mahony, 1995), or vice versa. This was evidenced by studieswhere levels of aroma compounds influenced panelist perception of sweetness andsourness for tomatoes (Baldwin et al., 1998), and levels of taste components influ-enced panelist perception of aromatic descriptors in mangoes (Malundo et al., 2001).

The volatiles in foods that can be perceived by the human nose are assumed tocontribute to the flavor of a food. Odor thresholds can be established (the level atwhich a compound can be detected by smell) as described by the Ascending Methodof Limits of the American Society for Testing and Materials, ASTM 1991 (Meilgaardet al., 1991). Log odor units can then be calculated from the ratio of the concentrationof a component in a food to its odor threshold. Compounds with positive odor unitsare likely to contribute to the flavor of a food (Buttery, 1989). This has been donefor tomato aroma compounds, for volatiles present at levels of one ppb or more(Buttery, 1993) (about 30 of the more than 400 identified compounds).

D

OES

F

RESH

-

CUT

Q

UALITY

I

NDICATE

F

LAVOR

Q

UALITY

?

Fresh-cut vegetable salads have great consumer appeal due to their convenience,flexibility of use and probably due to the fact that their desirable flavor often comesabout via condiments (croutons, spices or dressing), or because numerous productsmake up a medley mixture. Nonetheless, certain vegetables have specific characteristicaromas (mainly S-compounds) that must be perceived by the consumer. Consumeracceptance of fresh-cut fruits most often relies upon the inherent flavor and texturalquality of the product, seldom with accompaniments. Unfortunately, in the fresh-cutindustry, it is generally assumed that “if it looks good, it tastes good.” Slow marketgrowth for fresh-cut fruits may be attributed to the consumer’s apprehension to repeat-edly purchase products with inconsistent or unsatisfactory aroma and flavor quality.

Optimum harvest quality, postharvest quality and cutting quality are essentialfor maximizing fresh-cut shelf life. Harvest indices used for optimizing whole fruitshipping and or storage oftentimes should not be used for fruits destined to beprocessed. For example, an optimally mature load of cantaloupe may be shipped toa processor, received and then rejected because fruits are too soft or ripe for cutting.Additionally, controlled atmosphere (CA) stored apples with optimum visual appear-ance and firmness often have inferior aroma quality after long-term storage (Fellmanand Mattheis, 1995; Harada et al., 1985). Likewise,

Crucifers

stored under low O

2

may develop off-odors (Kaji et al., 1993; Lipton and Harris, 1974) that may notreadily dissipate after processing. The processor must, therefore, understand thephysiology of each commodity, their packaging and end product to judge accuratelywhen and what to process. Commercially, fresh-cut “quality” is generally only assessedvisually, and flavor quality is seldom assessed (aside from °Brix or acidity) beforeor after processing. Most fresh-cut research in the last decade focused on qualityretention based upon visual and subjective appearance and rapid common biochemicalanalyses. This approach (often guided by empirical observation) is often a physio-logically reliable tool for root and thick leafy tissues but is suspect regarding flavorof high water content fruits and vegetables. “Fresh-cut” should imply that a product

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is relatively fresh in terms of days since processing, and this should also helpsafeguard against inevitable flavor and aroma loss. Consequently, choice of variety,harvest condition, maturity, storage and shelf life with regard to flavor quality arebecoming active areas of research in fresh-cuts.

FLAVOR COMPOUNDS IN FRESH-CUT PRODUCE

The edible and fresh-cut portions of vegetables and fruits are derived from numerousbotanically different plant tissues. Subsequently, a plethora of compounds and com-pound classes may be important in fresh-cut flavor, depending upon which tissue(s)was used. For the most part, volatile compounds discussed in this chapter are thoseclassified as naturally occurring (endogenous). However, because fresh-cuts are “pro-cessed,” a discussion of secondary (reaction products) compounds is relevant. Secondarymetabolites may also have flavor contributions including bitterness, e.g., that relatedto sesquiterpene lactones in chicory (Peters and Amerongen, 1998), saltiness due tovarious natural salts, astringency related to flavonoids or alkaloids (DeRovira, 1997;Zitnak and Filadelfi, 1985) and tannins (Taylor, 1993). Natural flavor compoundsare intrinsic entities within a given tissue, whereas the secondary compounds aregenerally elicited products of enzymatic action (oxidative and hydrolytic degradationof lipids and their by-products) attributed to processing.

Although fresh-cuts are processed, and secondary compound production isinvoked, very little research has addressed the impact of secondary compounds uponfresh-cut flavor quality through storage. This is an interesting point, especiallyconsidering the fact that the act of mastication during consumption produces flavorsand characteristic flavor compounds in many commodities. For example, the char-acteristic flavor of garlic is due to 2-propenyl 2-propenethiosulfinate, however, thiscompound is only produced upon tissue rupturing (Carson, 1987). Many of thecharacteristic aromas in cabbage (

Crucifers

, in general), cucumber, green bean,tomato, olive, some melons, etc., are only produced after cutting, chewing or tissuedisruption. Because enzyme-mediated secondary compounds have been reported tobe both desirable and undesirable flavor compounds in edible plant products, it issafe to assume that a far greater quantity of “flavor” compounds in fresh-cuts mayin fact be “secondary compounds.” To date, little flavor and sensory work has beenperformed on fresh-cut fruits and vegetables. Two recent articles have reviewednumerous compounds considered to be important regarding flavor and aroma fornumerous fruits (Baldwin, 2002; Beaulieu and Gorny, 2002).

V

OLATILE

P

RECURSORS

AND

B

IOGENESIS

Flavor perception relies upon our sense of taste and smell. The tastes, generally due tosugars, organic acids and sometimes phenols, tannins and other minor compounds (e.g.,terpenoids and carotenoids), are “sweet,” “sour,” “bitter” and “salty.” In most fruits, acertain level of sugars (sweetness) or a balanced sugar-to-acid ratio determines consumersatisfaction. Oftentimes, the most important characteristic flavors for fruits and vegeta-bles are attributed to specific aroma compounds. The flavor volatiles for most commod-ities are complex, including a range of molecular weight alcohols, aldehydes, esters,

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ketones, lactones, sulfur-containing compounds and other compounds. Thus, numerousbiosynthetic pathways are responsible for flavor production in a given fruit or vegetable.

Various references will be made illustrating the likely biosynthetic pathwaysleading to many characteristic aroma and flavor compounds in fruits and vegetables.Numerous pathways involving different enzymes and substrates are involved andare regulated under vastly different physiological conditions. Therefore, we will notattempt to thoroughly cover genesis of flavor compounds for all fresh-cut commod-ities currently marketed, because it is out of the scope of this review. For schematicoverviews of flavor genesis, the reader is referred to the literature (Baldwin et al.,2000; Fellman et al., 2000; Sanz et al., 1997; Olías et al., 1993; Galliard et al., 1977;Yabumoto et al., 1977; Hatanaka et al., 1975).

Esters

Most fruits do not have characteristic aromas or flavors until they begin ripening. Asripening commences in climacteric melon fruit, methionine levels rise and ethylenesynthesis increases. Ethylene initiates and coordinates numerous diverse physiologicalpathways that essentially trigger a cascade of catabolic processes. Increased ethyleneproduction is often associated with an increasing free amino acid pool [for example,alanine, leucine, isoleucine, valine and methionine in muskmelons (Wyllie et al.,1996a)], membrane-mediated softening events, and as fruit maturity approaches, thisis accompanied by increased volatile production. The energy sources behind mostrespiratory events during ripening are sugars and organic acids, which are also highlyimportant concerning flavor, and sometimes reserve starch. Most primary aromacompounds are generated through

β

-oxidation of fatty acids, and secondary com-pounds are mainly formed as fatty acids that are oxidized via the lipoxygenase (LOX)pathway. As previously mentioned, secondary metabolites often have significant flavorroles in certain commodities. Although exhaustive information exists concerning fruitvolatile composition, there has been limited work performed on aroma biosynthesisand the relative contribution to flavor formation for the three main classes of aromaprecursors: amino acids, fatty acids and carbohydrates. Characterization of volatileand flavor formation and/or change occurring during preparation and storage of fresh-cuts is even more deficient.

Fatty acid and amino acid biosynthesis have long been shown to be importantin aroma biosynthesis (Drawert et al., 1973; Tressl and Drawert, 1973; Myers et al.,1970). In the 1960s, the typical flavor compounds in pears were believed to begenerated through

β

-oxidation of linoleic and linolenic acids (Jennings and Tressl,1964). Most unripe fruits synthesize and catabolize a variety of C

1

–C

20

fatty acidsand produce both primary and secondary alcohols. Together, these compounds areprecursors to one of the most important flavor and aroma compound classes in fruits—esters. In banana, simple amino acids such as [

14

C]leucine and [

14

C]valine were con-verted into the corresponding methyl-branched alkyl and acyl esters, alcohols andacids, whereas C

6

and C

9

aldehydes and C

9

and C

12

oxo acids resulted from oxidationof fatty acids (Tressl and Drawert, 1973; Myers et al., 1970). In apples, straight-chainfatty acid esters can be formed from

β

-oxidation of long-chain fatty acids, and thebranched-chain acid moieties arise from amino acids (Brackmann et al., 1993).

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Quantitatively speaking, fatty acids are the predominant precursors responsiblefor the characteristic flavor and odor volatile compounds in fruits. The first reactionactivating the

β

-oxidation spiral is when a saturated or unsaturated (Goodwin andMercer, 1988) fatty acyl CoA (even number of C atoms ) is oxidized via acyl CoAdehydrogenase. Each cycle through, and the final step of

β

-oxidation, produces acetylCoA, and a fatty acyl CoA, which is catalyzed by acetyl CoA acyltransferase.Subsequently, straight-chain acid backbone moieties for many esters are readilyavailable in plants, because their immediate precursors are intermediates of fattyacid

β

-oxidation (Lehninger, 1975; Conn and Stumpf, 1973).Ethyl esters share a common substrate (the ethyl moiety from ethanol), and the

acetate esters share another, the acetyl group (from acetyl-CoA), and both moietiescan be glycolytically derived from pyruvic acid (Yabumoto et al., 1978). Pyruvicacid is decarboxylated via pyruvate decarboxylase, forming acetaldehyde, which isreduced via ADH to ethanol. The oxidative decarboxylation of pyruvic acid by meansof coenzyme A (CoA) also yields acetyl-CoA, which is thought of as the directprecursor of esters and acetates. It is generally believed that an ester is enzymaticallyformed by combining an alcohol with an acyl group such as acetyl CoA (White et al.,1973; Forss, 1972).

The alcohol precursors for straight-chain ester biosynthesis are thought to bederived from oxidation of long-chain fatty acids through several cycles of the

β

-oxidation pathway resulting in a short-chain acyl CoA. Acyl CoAs are reduced tocorresponding aldehydes via acyl CoA reductase that is reduced further to the alcoholvia ADH (Bartley et al., 1985). In addition, research with deuterium-labeled precur-sors such as linoleic acid in apples showed that straight-chain esters are synthesizedvia the

β

-oxidation of fatty acids to give acetic, butanoic and hexanoic acids, whichmay be reduced to the corresponding alcohols before transesterification (Rowan et al.,1999). Furthermore, the use of deuterated precursors indicated that hexyl esters wereformed via a hexanoate intermediate, rather than hexanal (Rowan et al., 1999).

It is generally believed that low molecular weight branched chain esters (C

3

–C

12

)are synthesized by enzymatic combination of an amino acid and alcohol moiety(Yabumoto et al., 1977). Radio tracer techniques demonstrated that amino acids suchas leucine, isoleucine or valine were converted into branched-chain alcohols andesters in muskmelon. Valine was converted into esters containing the 2-methylpropylstructure (2-methylpropanoates), leucine was converted into 3-methylbutyl esters (3-methylbutanoates) and isoleucine was transformed into 2-methylbutanoates (isobu-tyrates) (Yabumoto et al., 1977). Apples infiltrated with L-isoleucine had increased2-methylbut-2-enyl and 2/3-methylbutyl esters (Hansen and Poll, 1993). Alanine wasfound to be the most important amino acid in terms of ester formation in strawberries(Pérez et al., 1992; Drawert, 1981). Alanine is an especially interesting amino acidin that it can supply both the ethyl group and acetate group found in many muskmelonaroma volatiles (Wyllie et al., 1995).

Aroma esters are believed to be synthesized enzymatically from alcohols andacyl CoA via alcohol acetyltransferase (AAT). AAT catalyzes the transfer of an acylmoiety from acyl-CoA onto the corresponding alcohol to form an ester. AAT is widelydistributed in fruits and has been investigated in apple (Fellman and Mattheis, 1995;Fellman et al., 1993a), banana (Ueda et al., 1992; Harada et al., 1985), melons

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(Ueda et al., 1997) and strawberry (Ke et al., 1994; Pérez et al., 1993). Many studiesinfer the presence of the enzyme via esterification of exogenous alcohol in fruit tissue;however, numerous cucurbit fruit lacked this enzymatic capacity (Ueda et al., 1997).Regardless, little work has been carried out to elucidate the properties of AAT, especiallyin fresh-cut products.

The first step in the conversion of an amino acid to an ester is deamination. Thisis followed by decarboxylation, various reductions and finally esterification (Pérezet al., 1992). The important enzymes in the conversion of isoleucine to 2-methylbutylesters and 2-methylbutanoate are

α

-aminotransferase,

α

-ketoacid decarboxylase,

α

-ketoacid dehydrogenase, ADH and AAT (Wyllie et al., 1996b). In banana, the con-centration of leucine and valine increased after the climacteric rise in respiration, asdid the corresponding branched-chain esters and alcohols (Tressl and Drawert, 1973;Myers et al., 1970). Based on the above examples, it is clear that a large proportionof aroma compounds contain structural elements derived from various amino acids(Wyllie et al., 1996b) and fatty acids.

Aromatic Compounds

Aromatic volatile compounds are formed chiefly by the amino acid

L

-phenylalanine.

D

-glucose is converted into phenolic compounds via the shikimic acid pathway, yieldingthe active precursor amino acid,

L

-phenylalanine. Glucose becomes phosphorylated andcondenses with phosphoenolpyruvate, and through a series of enzymatic transforma-tions, shikimic acid is derived, which is phosphorylated (via shikimate kinase) to 5-phosphoshikimic acid. 5-Phosphoshikimic acid is ultimately deaminated (via phenyla-lanine ammonia lyase, PAL) into

trans

-cinnamic acid and

trans

-

p

-coumaric acid, whichis further transformed into ferulic acid and sinapic acid. Cinnamic acid can undergoring substitution in a series of hydroxylation and methylation steps, resulting in variousacids that can be activated, as their corresponding esters of CoA. These activated esterscan enter various pathways leading to lignin, flavonoids, stilbenes, benzoic acids andother compounds (Schreier, 1984; Grisebach, 1981). The biosynthetic pathway forminglignin (through

p

-courmaric and ferulic acids) is responsible for various aromatic com-pounds such as alcohols, esters, flavonoids, hydroxyacids and amides (Gross, 1981).

Cinnamyl alcohols constitute the substrate pool leading to numerous characteristicaromatic compounds via side-chain elongations and degradations (Gross, 1981). Afterside-chain elongation, it is assumed that cinnamic acids proceed through

β

-oxidationto form benzoyl-CoA esters that could form benzoic acid esters or benzaldehyde andalcohols (Gross, 1981). Side-chain degradation, effectively removing an acetate unitfrom cinnamic acids, is proposed to be an important pathway resulting in the forma-tion of benzoic acids (Schreier, 1984). Although there are several different pathwaysfor the formation of benzoic acids, side-chain degradation of cinnamic acids is themost important mechanism (Gross, 1981). Characteristic aromatic compound pro-duction depends on the substitution pattern of the individual benzoic acids based ontheir respective phenylpropane precursors (Schreier, 1984). In tomato, phenylalaninecan be converted to 1-nitro phenylethane and then to phenylacetaldehyde

in vitro

bypH reduction (Buttery, 1993). [

14

C]phenylalanine was the precursor for phenylethanol,phenethylacetate, phenethyl butanoate and phenolic ethers in banana (Tressl and

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Drawert, 1973). The conversion of labeled phenylalanine into phenolic ethers suchas eugenol, eugenol methyl ether and elimicin was catalyzed by PAL, cinnamic acid-4-hydroxylase, phenolase and methyltransferase (Tressl and Drawert, 1973). Afterprocessing, increased PAL activity leads to browning and phenolic metabolism inlettuce (Ke and Saltveit, 1989). Browning-related phenolic oxidation compoundshave been implicated to contribute to off-flavors (Whitaker and Lee, 1995).

The mevalonic acid pathway forms terpenoids, carotenoids and geraniol (gera-nium oil). Aromatic terpenes are generally considered secondary defense com-pounds, yet some such as limonene and menthol have aroma attributes as well. Theoxygenated terpenoid volatiles linalool, neral and geranial, which are important forflavor in some fruits, have been identified in ripe tomatoes (Buttery and Ling, 1993).Lactones that are important flavors of peaches and apricots are produced via LOXactivity (Crouzet et al., 1990). Extensive information concerning formation of chiral

δ

-lactone and

γ

-lactone and the pathways has been published (Tressl et al., 1996).Certain aromatic compounds are believed to be formed as breakdown products

from various aromatic pigments such as lycopene, carotene, etc., and these compoundsapparently occur as oxidation products as well (Buttery, 1981). For example,

β

-iononeprobably results from the oxidative breakdown of

β

-carotene. Therefore, during wound-ing or processing (cutting or chewing), the induction of certain volatiles (predominatelysecondary metabolites or oxidation products) may affect flavor attributes, especiallyin fruits and vegetables with high concentrations of readily oxidizable aromaticpigments (i.e., carotene) and fatty acids.

Aldehydes and the Lipoxygenase Pathway

The pleasant odor in cucumber was attributed to 2,6-nonadienal, and two unsaturatedaldehydes (2-hexenal and 2-nonenal) and three saturated aldehydes (ethanal, propanaland hexanal) were considered to contribute secondarily to overall flavor (Forss et al.,1962). Using cucumber homogenates radiolabeled with

14

C-linolenic (18:3) and

14

C-linoleic (18:2), the flavor active aldehydes propanal (

E

) 2-hexenal and (

E

,

Z

)2,6-nonadienal were related to linolenic acid, whereas hexanal and (

E

) 2-nonenalwere related to linoleic acid (Grosch and Schwarz, 1971). However, cutting ormechanically rupturing cucumber fruit tissue was associated with enzymaticallyproduced aldehydes (Fleming et al., 1968). Production of green-apple-like odors,known to be related to hexanal and (

E

) 2-hexenal, increased upon crushing or cuttingfruit cells (Drawert et al., 1966). C

6

aldehydes and alcohols have since been recoveredafter tissue disruption in numerous crops (e.g., apple, banana, grape, green leafytissue, olive and tomato) and are related to the LOX pathway.

Lipoxygenase (Galliard and Phillips, 1976), a hydroperoxide lyase (HPL)(Galliard et al., 1976) and (

Z

)-3: (

E

)-2-enal isomerase (Phillips et al., 1979) enzymesare involved in the formation of volatiles from fatty acid precursors. The LOXpathway is also responsible for production of C

6

aroma compounds [i.e., (

Z

)-3-hexenal, (

Z

)-3-hexenol and (

Z

)-3-hexenyl acetate] in green leafy and fruit tissue(Gardner, 1989). Acyl hydrolases (AH) effectively catalyze the hydrolysis of esterbonds in monoglycerides, monogalactosyldiglycerides, lysoglycerophospholipides,diglycerides, digalactosyldiglycerides and glycerophospholipids but not ester bonds

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in triglycerides. AH enzymes are very active and destroy most membrane-boundpolar lipids that lead to the free fatty acid pool (C

16:0

, C

18:2

, C

18:3

) when tissue isdisrupted (Goodwin and Mercer, 1988). Lipid AH breaks down linolenic acid, whichis hydroperoxygenated by LOX into hydroperoxylinolenic acid, then HPL cleavesthis into (

Z

)-3-hexenal and 12-oxo-(

Z

) dodecenoic acid. The (

Z

)-3-hexenal is sub-sequently reduced via ADH to (

Z

)-3-hexenol, which is then esterified to (

Z

)-3-hexenyl acetate (Hatanaka, 1993; Anderson, 1989; Sekiya et al., 1982). The LOXand HPL enzyme system involved is bound to the thylakoid membrane of chloro-plasts in green leaves. Lipoxygenase adds oxygen stereoselectively to unsaturatedfatty acids having a (1

Z

, 4

Z

)-pentadiene moiety (e.g., alpha-linolenic and linoleicacids), to produce 13-(

S

)-hydroperoxides that are next cleaved by HPL at the bondbetween C

12

and C

13

of these hydroperoxides to form C

6

aldehydes (Hatanaka, 1993).“Green” and “grassy” food flavors are generally due to C

6

compounds formedfrom unsaturated aliphatic C

18

fatty acids oxidized by LOX and intermediary substratesconverted into various organoleptic compounds via HPL. For example, the charac-teristic tomato flavor compounds hexenal and (

Z

)-3-hexenal are thought to be sec-ondary compounds (Riley and Thompson, 1998). Likewise, the characteristic flavorcompounds of bell peppers [hexanol, hexanal, (

Z

)-3-hexen-1-ol, (

E

)-2-hexenal, (

E

)-2-hexen-1-ol] and cucumbers [(

E

,

Z

)-2, 6-nonadienal, (

E

)-2-nonenal and 2-hexenal]are generated enzymtically via LOX, as a consequence of cutting or homogenization(Matsui et al., 1997; Wu and Liou, 1986; Fleming et al., 1968). LOX and HPL havebeen found in many fruits commonly fresh cut (Pérez et al., 1999b; Matsui et al., 1997;Riley et al., 1996; Olías et al., 1993; Kim and Grosch, 1981; Vick and Zimmerman,1976). In bell peppers, both HPL and LOX activities decreased with maturation, andthe amounts of C

6

aldehydes and alcohols formed from homogenization of mature fruitalso decreased (Matsui et al., 1997). Subsequently, selection for varieties with lowLOX and HPL activity may be critical in crops where cutting may provoke off-flavors.

Hexyl acetate arising from olive crushing has been associated with lipid degrad-ing enzymes as well as a complete enzyme system leading to green odor notes (Olíaset al., 1993). Triacylglycerols and phospholipids (mainly polyunsaturated) are hydro-lyzed by AH. LOX then cleaves the resulting linoleic and linolenic acids into 9- and13-hydroperoxides, and then HPL selectively cleaves the 13-hydroperoxide to formhexanal and (

Z

)-3-hexenal. Hexanal and hexenal (oftentimes including isomerizations)are reduced via ADH to their corresponding alcohols. Finally, AAT, with the partici-pation of acetyl-CoA (hence, not a direct esterification), produces an ester from thealcohol (Olías et al., 1993; Pérez et al., 1993). Similar results concerning esterformation (oftentimes utilizing precursor feeding regimes) via LOX and

β-oxidation,dependent upon available acetyl CoA, have been reported in apple (Rowan et al., 1996,1999; Berger and Drawert, 1984), banana (Harada et al., 1985; Myers et al., 1970)and strawberry (Yamashita et al., 1975).

IMPORTANT AROMAS AND FLAVORS

Although wounding tissue invokes secondary aldehyde and alcohol production, manyaldehydes have been considered to impart characteristic and desirable odors to foodsso long as their concentrations are extremely low. Furthermore, cell disruption is

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400 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

sometimes necessary to allow enzymes and substrates that were formerly compart-mentalized to interact (Buttery, 1993). For example, some aroma compounds arebound to sugars as glycosides (celery, lettuce) or glucosinolates (cabbage, radish).Glycosidically bound furaneol (2,5-dimethyl-4-hydroxy-2H-furan-3-one) and itsmethyl ether mesifurane are important aroma components in strawberry that appearto have D-fructose as a precursor (Pérez et al., 1999a; Zabetakis et al., 1996). Thislinkage can be cleaved by enzyme action or heat (cooking). Bound volatiles werealso found in fruits such as apricot, mango, grape and passion fruit (Chassagne andCrouzet, 1995). Others are breakdown products of lipids, amino acids, lignin orpigments (Buttery and Ling, 1993). Secondary metabolites can also be produceddue to wounding of tissue that occurs during processing (Wong, 1994). For example,there is an accumulation of glycoalkaloids in damaged potato tubers and an increasein polyphenols in many tissues that brown on the cut surface, such as apples andpears, for which the flavor impact is unknown.

Thioesters have been reported to be probable flavor notes responsible for the“earthy” and “musky” notes in muskmelon (Wyllie et al., 1994). In addition, methioninecan be converted into thioether esters (Wyllie et al., 1995). Glucosinolates (thioglu-cosides) are naturally occurring secondary plant metabolites that have been foundin the Cruciferae family. These metabolites (mainly sulfur containing) are responsiblefor the taste and odor, termed “mustard oils,” in these vegetables (Ju et al., 1982),especially upon cell rupture. When Cruciferae cells are ruptured, glucosinolates undergoenzymatic hydrolysis with the endogenous myrosinase enzymes (thioglucosidase),releasing thiocyanates, isothiocyanates (Wattenberg, 1978) sulfate and glucose(Ju et al., 1982). Therefore, extra precautions must be taken to insure that off-odorsand off-flavors do not jeopardize the marketability of shredded Crucifer products.

SUGARS (SOLUBLE SOLIDS), ORGANIC ACIDS AND TITRATABLE ACIDITY

Important flavor contributions for many fruits and vegetable are attributed to specificorganic acids and various sugars. Glucose, sucrose and fructose are the most impor-tant sugars that affect the perception of sweetness. Of these, glucose is perceivedas less sweet and fructose more sweet than sucrose. Thus, a weighting of these sugarsin relation to sucrose and subsequent combining can give a single value “sucroseequivalent” (SE) for a sample (Koehler and Kays, 1991). Sugars are commonly thoughtto be synonymous with soluble solids (SS). However, the real proportion of sugarsmeasured in SS depends on the fruit or vegetable, and this may still not indicate clearlythe contribution to flavor. For example, in orange, SS appear to relate to sweetness,while in tomato and mango, the relationship is not clear (Malundo et al., 2001; Baldwinet al., 1998, 1999a). It is possible that cut produce that exhibits high respiration dueto wounding may catabolize sugars or acids as a carbon source during storage. Sweet-ness, flesh firmness and taste are very important characteristics for fresh-cut melonquality. It is a well-established fact in the food industry that sugar content is generallypositively correlated with desirable flavor quality. However, occasionally, too muchsugar can be perceived negatively. The best sugar range for storage of fresh-cutcantaloupe was found to be 10–13°Brix, however, some people judged the fruit as

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Flavor and Aroma of Fresh-cut Fruits and Vegetables 401

being too sweet at 13°Brix (Anonymous, 2000). In 17 Western cantaloupe varieties,there was an average 5% decrease in SS content (range 0–11%) and an average8% decrease in sugar concentration (range 0–21%) when cubes were stored 12 days(in air) at 5°C (Cantwell and Portela, 1997). Likewise, SS were found to decreasein cut peaches during storage at 2°C in either air or 2% O2, which affected flavor(Mencarelli et al., 1998). °Brix decreased 9.7% (range 2.3–13%) in cantaloupe ballsprepared from four Eastern varieties stored 8 days at 0°C (Lange, 1998). However,CA-stored melon pieces had higher SS concentrations than air-stored (10.3% vs.9.5% and 10.2% vs. 9.1%, at 10°C and 5°C, respectively), after 9 days at 10°C and15 days at 5°C (Cantwell and Portela, 1997). Soluble solids remained somewhatconstant for 7 days storage (4°C) in fresh-cut cantaloupe when harvest maturity wasat least half-slip, but rapidly declined after only 5 days of storage in quarter-slipcubes (Figure 12.1). Cubes prepared from fruit harvested at quarter-slip had signif-icantly lower (0.05 level) °Brix compared to all other harvest maturities on all days,except day 0. However, in fresh-cut ‘Gala’ apple, sugars remained constant during14 days of storage at 1°C, although sweetness and sweet aromatic flavor increasedthen decreased (Bett et al., 2001).

Titratable acidity and pH have also been used to assess the sugar-to-acidity ratioin some fresh-cut fruits. Changes in TA, pH and SS in apple slices from 12 cultivarsthat were stored at 2°C for 12 days were small and varied by cultivar (Kim et al.,1993). In cut ‘Gala’ apple, pH decreased during 2 weeks storage at 1°C, while%TA increased then decreased and sensory perception of sourness also fluctuated

FIGURE 12.1 °Brix change (Atago PR-101) in fresh-cut cantaloupe prepared from fruitharvested at different maturities, held at 4°C (n = 30). All quarter-slip means, except day 0,were significantly different (0.05 level) from other maturities according to an analysis ofvariance performed with Duncan’s multiple-range test.

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402 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

(Bett et al., 2001). Fresh-cut persimmons stored in CA for 7 days at 5°C hadincreased SS for 3 days then decreased by day eight, and pH tended to increasethrough storage (except when stored under 2% O2) (Wright and Kader, 1997). A17% loss in SS and a twofold acidity increase occurred after only 2 days of storageat 20°C in cantaloupe slices, but acidity change was attributed to lactic acid bacteria(Lamikanra et al., 2000). Titratable acidity in fresh-cut oranges stored at 4°C for8 days decreased 36% (Rocha et al., 1995).

The main organic acids regarding flavor notes for most fruits are malic, citric,tartaric, succinic and quinic acids (Kays, 1997). Sugars are the primary product ofphotosynthesis, whereas most organic acids are synthesized from glycolytic precursorsin the tricarboxylic acid pathway as products of respiration. A characteristic flavoris often attributed to a high concentration of one organic acid (e.g., citric acid inlemon) or to the overall acidity or sugar-to-acid ratio inherent to the product/variety(e.g., tomato and apple). Acids such as citric in citrus and tomatoes, tartaric in grapesand malic in apples, give fruit and vegetables their sour flavor. Some fruits, likemelon or banana, have very little acid (Wyllie et al., 1995). Sometimes, measurementof SS, the ratio of SS/TA or pH relate better to sourness than TA itself (Malundo et al.,2001; Baldwin et al., 1998).

FACTORS AFFECTING FRESH-CUT FLAVOR

Overall, flavor is affected by genetics, preharvest environment (Baldwin et al., 1995c;Kim et al., 1993; Romani et al., 1983), cultural practices (Romig, 1995; Wright andHarris, 1985), harvest maturity and postharvest handling or storage (Baldwin et al.,1999a,b; Gorny et al., 1998a; Maul et al., 1998a,b; Watada et al., 1996; Mattheis et al.,1991, 1995; Fellman et al., 1993b). Generally speaking, flavor of fresh produce willnot improve after harvest (aside from the effect of continued ripening in climactericfruit), and therefore, flavor deterioration should be minimized. This is an especiallydifficult task in fresh-cut products, where biochemical changes due to wounding canaffect shelf life and flavor quality. Processing technique (Bolin et al., 1977; Saltveit,1997; Wright and Kader, 1997), sanitation (Hurst, 1995), packaging (Cameron et al.,1995; Solomos, 1994) and temperature management during shipping, handling andmarketing (Brecht, 1999) also play important roles in maintenance of fresh-cutquality.

Physical alterations and potential low O2 atmospheres in packages may createsignificant negative changes in flavor and aroma. There are also synergistic interactionsbetween numerous factors such as variety, source, season, initial maturity, optimumprocessing maturity, slicing and cutting equipment, GRAS treatments, container orbag [including modified atmosphere packaging (MAP)], temperature management,shipping, handling and length of shelf life. The synergistic interaction between theabove factors may have negative consequences on flavor attributes and sensoryacceptability. However, little information is available regarding the aggregate effectsthese factors have on flavor quality. Therefore, properly preparing, packaging andhandling fresh-cut products is essential to avoid potential flavor loss or change thatmay actually cause a decrease in consumer satisfaction.

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Flavor and Aroma of Fresh-cut Fruits and Vegetables 403

MORPHOLOGICAL CONSIDERATIONS

Fruit tissues, due to their unique anatomical nature, are very susceptible to bruisingand mechanical injury. This is very different from most fresh-cut vegetables thatmay be derived from very durable root tissues (e.g., carrots, radishes) or pliable leaftissues (e.g., iceberg lettuce, cabbage). Because a commodity’s natural cuticle orskin barrier to gas diffusion is compromised during processing, accelerated flavorvolatile loss most likely accompanies increased respiration and ethylene production.

CHILLING INJURY AND STORAGE TEMPERATURES

One type of chilling injury (CI) is the loss of aroma compounds. Chilling tomatofruit to 5°C for one week with subsequent ripening at 20°C affected the fruit flavor(Kader et al., 1978). In other studies, tomato fruit stored at 2, 5, 10 and 13°C wereshown to have reduced levels of important volatiles (Maul et al., 2000; Buttery et al.,1987). Tomatoes stored at 2, 5, 10 or 12.5°C were also shown to have less ripearoma and flavors as well as more off-flavors compared to fruit stored at 20°C bya trained descriptive panel (Maul et al., 2000). Cut tomato must be stored at 5°C orlower to prevent spoilage. If not ripened properly, >27–49 Newtons (N), prior tocutting, stone fruit are susceptible to aberrant ripening that negatively affected eatingquality when fresh-cut product was stored at CI temperatures (Gorny et al., 1999a).A significant number of fruit are CI sensitive as whole intact fruit before processing.Examples include fruit such as pineapple, cantaloupe, honeydew, watermelon, peach,nectarine and mango. Therefore, in certain cases, chilling temperatures before pro-cessing and subsequent holding of fresh-cut products at low temperatures may havenegative consequences on flavor quality. Intermittent warming and brief heat treat-ments have been employed in order to alleviate CI in some crops. However, heattreatment of apple fruit to reduce physiological and pathological disorders inhibitedemission of volatile esters important to apple flavor (Fallik et al., 1997). Furthermore,commercially harvested ‘Gala’ apples that were heat treated after harvest and latersliced and stored showed lower volatile levels than unheated controls (Table 12.1).

VARIETIES, GROWING REGION AND SEASON

Preharvest factors such as sunlight, water availability, fertilization and chemical appli-cations undoubtedly affect the condition of the crop. This, in turn, can have an effecton the internal quality characteristics of the harvested product, including flavor.Preharvest treatment with aminoethoxyvinylglycine (AVG) suppressed volatile pro-duction in pears by approximately 50%, while ethylene exposure reversed the sup-pression (Romani et al., 1983). Heavy rains prior to harvest appeared to dilute flavorcompounds in tomato (Baldwin et al., 1995a). Fruit from tomato plants treated withincreased levels of nitrogen and potassium fertilizer scored lower in sensory analysisand showed increased levels of TA, SS and several volatiles (Wright and Harris,1985). Greenhouse-grown tomatoes exhibited lower levels of numerous volatile com-ponents compared to their field-grown counterparts (Dalal et al., 1967). Accordingto a trained sensory panel, mite control prior to harvest resulted in field-grown

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404 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

strawberries with more sweetness and flavor intensity than those receiving no treat-ments (Podoski et al., 1997). Varieties of fruits and vegetables have been shown todiffer in flavor based on sensory and chemical analysis, reflecting their geneticdiversity. For example, aroma in apples was not the result of the same compoundsin every cultivar, although some volatile compounds that seemed to be importantwere common to all 40 cultivars studied (Cunningham et al., 1985). Varieties performoptimally in certain growing regions and often have variable postharvest qualityattributes depending on cultural practices, climate, season and harvest maturity. Theproportions of dominant apple volatiles varied by season (López et al., 1998), anddesirable pineapple flesh volatile oil content was higher in summer fruit than inwinter fruit (Haagen-Smit et al., 1945). Several reports have documented clearly thatcertain cultivars outperform others with regard to fresh-cut shelf life and keeping quality(Anonymous, 2000; Gorny et al., 1998b, 1999b; Lange, 1998; Cantwell and Portela,1997; Kim et al., 1993). However, little to no data are available concerning flavorand sensory quality for fresh-cuts produced from different varieties grown underdifferent cultural conditions.

Harvest maturity can also affect the flavor of the ripened product. This isespecially important for fresh-cut produce, where harvest maturity can also affectthe shelf life of the product. Ideally, horticultural products are harvested at a stagethat gives optimal eating quality. In reality, this optimal quality is often sacrificedto minimize physical damage during shipping, handling and processing to maximizeshelf life. Harvest maturity affected ester formation in apples, depending on exactclimacteric stage at time of harvest (Fellman et al., 1993a). Acid levels decreasedas days after full bloom increased for apple, and this affected sensory responses for

TABLE 12.1Effect of Heat-Treating Intact Commercially Harvested ‘Gala’ Apple Fruit on Important Volatile Components in Fresh-cuts during Storage at 8°C. Intact Apples were Subjected to 38°C at 98% RH for 4 Days Prior to Slicing

Day 0 Day 7

Volatile Compound (ng g−1) Control Heat Control HeatButanol 429 a1 73 c 200 b 60 cButyl acetate 499 a 14 c 148 b 17 cButyl butanoate 4 a 5 a 0 c 1 bButyl hexanoate 3 a n.d.2 1 b n.d.2-Methylbutyl acetate 15 a 4 b 4 b 3 b2-Methylbutyl 3-methylbutanoate 4 a 3 a 3 a 3 aHexanal n.d. 7 b n.d. 14 aTrans-2-hexenal 5 a 4 a 6 a 5 aHexyl acetate 19 a 1 c 5 b 1 c

1 Mean values (n = 3) in the same row that are not followed by the same letter show significantdifference (p < 0.05).

2 n.d. = not detected.

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Flavor and Aroma of Fresh-cut Fruits and Vegetables 405

tartness (Plotto et al., 1997). However, apples harvested later were found to be morefruity and sweet compared to apples harvested two weeks earlier (Cliff et al., 1998).Harvest maturity was shown to affect both the sensory and chemical analysis ofripened tomato fruit (Maul et al., 1998a). Tomatoes harvested at the immature greenstage resulted in ripened fruit with lower volatile levels than mature green-harvestedtomatoes, while tomatoes harvested table ripe displayed higher intensities for sweet-ness, saltiness and fruity floral aroma (due to levels of both volatile and nonvolatilecomponents) than green or breaker-harvested fruit (Watada et al., 1979). Similarly,fruit harvested at the turning-red stage were sweeter, less sour and more tomato-like, with less off-flavor than earlier-harvested fruit (Kader et al., 1977). Harvestmaturity also affected consumer acceptability ratings of mango and trained descrip-tive panel ratings for sweetness, sourness and various aroma descriptors. Fruitharvested later were sweeter, less sour and generally had more intense aroma char-acteristics (Baldwin et al., 1999a). It is not difficult to assume that these findingsfor intact fruit would apply to the fresh-cut product as well.

RIPENESS AT CUTTING, FIRMNESS AND PROCESSING

Many fruits are picked before they are fully ripe. Therefore, the question arises asto what maturity should climacteric fruit be when fresh-cut in order to optimizeproduct shelf life and eating quality? Both the maturity at harvest and the ripenessstage at cutting will affect the postcutting quality and shelf life of fresh-cut fruitproducts. Mature-green tomato fruit ripened normally and attained comparable eat-ing quality compared to those fruit that were sliced after the whole fruit ripened(Mencarelli and Saltveit, 1988). However, little research has addressed whethernormal ripening will continue in other climacteric fruit if the cutting process is doneon unripe fruit. In immature sliced pear and peach fruit, softening occurred, butother ripening-related processes such as flavor development and texture seemedaberrant when fruit were processed at an excessively immature stage (Gorny et al.,2000; Beaulieu et al., 1999; Gorny et al., 1998a; Mencarelli et al., 1998).

Maturity at cutting for many fruits can help to predict potential flavor qualitythrough storage. For example, the optimum initial fruit firmness for ‘Bartlett’ pears,for maintenance of firmness without browning in fresh-cut slices, was found to beroughly 49 N (Dong et al., 2000; Sapers and Miller, 1998). Fresh-cut pear slicesprepared from firm ‘Bartlett’ and ‘Bosc’ fruit (70–85 N) were excessively firm andlacked flavor (Dong et al., 2000). For climacteric fruits, initial fruit firmness may,therefore, be a good indicator of fruit ripeness for optimum postcutting flavor quality.A mature green cantaloupe will not have sugars or volatiles associated with a desirableripe fruit (Pratt, 1971). Melon fruit harvested before fully ripe (full-slip) developed onlyabout one quarter the total volatiles as compared to three-day-old fully ripe fruit (Wyllieet al., 1996a). Mature green cantaloupe (i.e., <half-slip) will deliver a fresh-cut productthat has optimum visual shelf-life, but sugars (Figure 12.1) and volatiles (Beaulieu andGrimm, 2001) will be severely compromised, and this trend was conserved through 10days fresh-cut storage at 4°C. As harvest maturity increased, the relative amount (SPME,GC-MS) of 29 esters increased, yet, quarter-slip fruit had roughly one-third the vola-tiles as compared to full-slip fruit (Figure 12.2). After five to seven days storage,

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volatiles declined slightly in cubes that were prepared from >half-slip maturity fruit.These data may indicate that harvest maturity is critical for fresh-cut flavor quality.

CHEMICAL AND PHYSICAL TREATMENTS

Many physical and chemical treatments have been applied to whole produce, andthe efficacy of some of these treatments is currently being investigated on fresh-cuts. However, as with numerous aforementioned protocols, very little aroma orflavor work has been accomplished where edible coatings, disinfection, natural plantproducts, ethylene absorbents, gamma irradiation, heat shock, microbial competitionand pulsed-microwave irradiation have been used on fresh-cuts. For more physiologicaldetails and discussion regarding the use of treatments geared toward extendingacceptable fresh-cut quality, please see Chapter 9.

Chlorination and Washes

Chlorination, as commonly used for fresh-cut salad sanitation, may not be desirablefor all fresh-cut fruits. Postcutting washing and/or dipping may have negative conse-quences regarding increased water activity and the “washing away” of desirable flavorattributes. Numerous processors do not wash freshly cut fruits that have little or nobrowning (e.g., melons and strawberry), because GRAS treatments are seldom appliedand because water removal (centrifugation or spinning) can severely damage the tissue.

Calcium Salts and Antibrowning

Application of aqueous solutions of calcium salts, ascorbate, citric acid, isoascorbicacid and sodium erythorbate (generally 0.5–1.0% solutions/dips) can help maintain

FIGURE 12.2 Change in total esters recovered (solid-phase microextraction and gas chro-matography-mass spectrometry; SPME, GC-MS) from fresh-cut cantaloupe prepared withfruit harvested at four maturities, stored at 4°C (n = 3 ± standard deviation).

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fresh-cut tissue firmness and reduce surface browning (Gil et al., 1998; Gorny et al.,1998b; Izumi and Watada, 1995; Rosen and Kader, 1989; Sapers and Zoilkowski,1987; Morris et al., 1985; Ponting et al., 1971, 1972). Unfortunately, some treat-ments that reduce enzymatic browning or improve texture can impart off-flavors.For example, calcium chloride has been shown to impart a detectable off-flavor incantaloupe slices at concentrations above 0.5%, whereas calcium lactate improvedfirmness without imparting bitter flavor (Luna-Guzmán and Barrett, 2000). After10 days of storage in air at 0°C, 70% of consumers judged fresh-cut ‘Bartlett’ pearthat were treated with 2% ascorbic acid, 1% calcium lactate and 0.5% (w/v) cysteineto have acceptable flavor that was undistinguishable from the controls (Gorny et al.,2002).

Numerous new experimental compounds (ascorbic acid-2-phosphate and ascor-bic acid-2-triphosphate, calcium propionate, cysteine, N-acetylcysteine and 4-hex-ylresorcinol) are being tested for antibrowning capacities. Yet, we are unaware whateffects most of these compounds will have upon flavor and aroma in fresh-cuts. Itis suspected that 4-hexylresorcinol may impart an unacceptable off-flavor on fruitproducts. ‘Red Delicious’ apple slices treated with a combined antibrowning dip (4-hexylresorcinol, isoascorbic acid, N-acetylcysteine and calcium propionate) held at5°C maintained visual quality for five weeks, yet microbial decay was evident afterfour weeks (Buta et al., 1999). Analyses of organic acids and the major sugars revealedthat the slices treated with combined antibrowning compounds retained higher levelsof malic acid and had no deterioration in sugar levels at 5 and 10°C, indicating thathigher quality was maintained during storage. One would expect that these resultsshould also translate into maintenance of flavor quality.

Antimicrobial, Edible Coating and Other Treatment Compounds

Treatment of fruits and vegetables with acetaldehyde, ethanol or low O2 (which canresult in production of acetaldehyde and ethanol) has resulted in flavor enhancementof pears, tomatoes and blueberries (Paz et al., 1982), grapes (Pesis and Frenkel, 1989),strawberry (Pesis and Avissar, 1990) and feijoa (Pesis et al., 1991). For example,in strawberry, an increase in acetaldehyde, ethanol, methyl acetate, ethyl acetate andethyl butyrate was found after application of acetaldehyde (Pesis, 1996). Treatmentof oranges with acetaldehyde resulted in induced synthesis of ethylbutyrate (Shawet al., 1991). Treatment of tomatoes, blueberries and pears led to enhanced sensoryquality, in part due to increased sugars (Paz et al., 1982), and reduced acidity in figand orange (Pesis and Avissar, 1989; Hirai et al., 1968). ‘Red Delicious’ apples werestored in an atmosphere containing ethanol vapors for 24 hours, which resulted ina threefold increase in ethyl ester concentrations (Berger and Drawert, 1984). However,attempts to promote volatile and sensory attributes with vaporous acetaldehyde oralcohol in fresh-cut fruits may be quite challenging, because these compounds havea maturity- and concentration-dependent effect on inhibiting or promoting ripening(Beaulieu and Saltveit, 1997; Beaulieu et al., 1997).

Nevertheless, treatment of whole fruit with acetaldehyde, ethanol or low O2 mayimprove flavor of the subsequent fresh-cut product. In addition, acetaldehyde and ethanolhave antimicrobial properties. For example, acetaldehyde reduced Botrytis cinerea

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and Rizopus stolonifer on strawberries and grapes (Prasad and Stadelbacher, 1974).Whole apples were treated with ethanol vapor prior to slicing, and sliced applesreceived an ethanol dip. In both cases, after two weeks of storage, the ethanol-treatedapples were higher in some volatiles, including several esters (Table 12.2).

Hexanal is a natural aroma precursor in apples that is readily converted to aromavolatiles in vivo by fresh-cut apple slices (Song et al., 1996). Hexanal not onlyenhanced the aroma of fresh-cut apple slices, but it also reduced enzymatic browningat the cut surface as well as inhibited molds, yeasts and mesophilic and psychrotrophicbacteria in ‘Ganny Smith’ slices stored at 15°C (Lanciotti et al., 1999). Research iscurrently in the initial stages for the use of this compound on fresh-cut fruit products.However, it is currently not approved for use.

Microbial spoilage of cut fruit products can affect their flavor quality and shelflife. Methyl jasmonate is a volatile, naturally occurring compound found in manyplants and has been reported to have hormone-like activity at very low concentrations.Exogenously applied methyl jasmonate has been shown to be very effective inreducing mold growth on fresh-cut celery and peppers and may, in the future, haveapplications as a naturally derived fungicide (Buta and Moline, 1998). Citric acidis a GRAS-listed compound that is a natural organic acid and can be used as apreservative, acidulant or flavoring agent in foods. By acidifying the surface of cutproducts, citric acid can reduce the microbial load and thus improve flavor. The shelflife of peeled oranges was extended by 0.5–1.0% citric acid infusion treatments that

TABLE 12.2Effect of Exogenous Ethanol on Ethyl Ester and Other Major Volatile Ester Contents in Fresh-cut ‘Gala’ Apples during Storage at 8°C. Intact Fruits were Incubated in Saturated Ethanol Vapor at 23°C for 24 Hours before Slicing, or Cut Slices were Dipped in 70% Ethanol for 30 Seconds

Day 0 Day 7

Esters (ng g−1) Control Vapor Dip Control Vapor DipEthylesters

Acetate 22 e1 3386 a 145 d 1032 c 2437 b 2585 bPropanoate 521 d 2912 c 9986 a 865 d 2270 c 5682 bButanoate 2 c 5 b 1 c 148 a 110 a 121 a2-Methylbutanoate 2 c 11 b 1 c 21 a 16 a 22 aHexanoate 2 b 4 a 2 b 2 b 2 b 2 b

Other major estersButyl acetate 535 a 240 c 399 b 158 d 104 e 63 fButyl butanoate 4 b 10 a 5 b 2 c 3 c 2 cButyl hexanoate 4 b 12 a 6 b 2 c 3 c 3 c3-Methylbutyl 2-methylbutanoate 0 b 2 a 0 b 0 b 0 b 1 aHexyl acetate 19 a 16 a 17 a 5 b 4 b 3 b

1 Mean values (n = 3) in the same row that are not followed by the same letter show significant difference(p < 0.05).

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inhibited growth of spoilage bacteria (Pao and Petracek, 1997). Diced celery flavorand shelf life were improved by treatment of samples with 0.5 and 1.0 kGy gammairradiation compared to conventional treatments such as acidification, blanching andchlorination (Prakash et al., 2000). Although all samples lost flavor over 22 days ofstorage, irradiated samples maintained color, texture and aroma longer than samplesfrom other treatments and had less off-flavor. Sensory shelf life was extended 7–29 days,in part due to reduced aerobic microbial plate counts. 1-Methylcyclopropene (MCP)has been shown to block ethylene action and, thus, inhibit many ethylene responsessuch as ripening, softening, etc., as has been shown on avocados (Feng et al., 2000),apricots (Fan et al., 2000) and bananas (Harris et al., 2000). The effects of MCPcould be useful for fresh-cut products. Whole apples treated with MCP and latersliced and stored were firmer but contained less aroma compounds than nontreatedfruit after one week (Table 12.3).

Use of edible coatings can improve fresh-cut fruit quality. Peeled packaged citrusproducts have a shelf life of approximately 17–21 days, but fluid leakage can beproblematic. Edible wax microemulsion coatings (up to 12% solids) reduced leakageof dry-packed grapefruit segments by 80% after 2 weeks and 64% after 4 weeks ofstorage (Baker and Hagenmaier, 1997). Coatings with carnauba wax were found tobe most effective, and coatings were not detected by informal taste panels beforeor after storage (Baker and Hagenmaier, 1997). Cut apples, treated with acidiccoatings, exhibited lower microbial populations, without an overly acidic flavor(Baldwin et al., 1996). Research on use of edible coatings on fresh-cuts has recentlyescalated, but little work has been published concerning retention of flavor quality.

TABLE 12.3Effect of Treating Intact ‘Gala’ Fruit with 1-Methylcyclopropene (MCP) on Major Volatile Contents in Fresh-cut Apples during Storage at 8°C. Treatment Consisted of 1 ppm MCP for 18 Hours at 20°C prior to Slicing

Day 0 Day 7

Volatile Compounds (ng g−1) Control MCP Control MCPButanol 201 b1 296 a 100 c 73 dButyl acetate 323 b 392 a 143 c 90 dButyl butanoate 5 a 5 a 1 b 1 bButyl hexanoate 2 a 3 a 1 b n.d.2

2-Methylbutyl acetate 28 a 29 a 3 b 3 b2-Methylbutyl 3-methylbutanoate 4 a 3 a 3 a 3 aHexanal n.d. 6 a n.d. n. d. Trans-2-hexenal 6 b 9 a 5 b 2 cHexyl acetate 15 a 16 a 6 b 2 c

1 Mean values (n = 3) in the same row that are not followed by the same letter show significantdifference (p < 0.05).

2 n.d. = not detected.

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CONTROLLED ATMOSPHERE, MODIFIED ATMOSPHERE PACKAGING AND FLAVOR

The beneficial effects of CA storage for whole fruits and vegetables have been welldocumented, and CA storage is widely employed throughout the produce industry.However, CA storage alters the flavor of apples (Yahia, 1994), and if prolonged,reduces the volatile emission compared to air-stored fruit (Fellman et al., 1993a;Yahia, 1991; Lidster et al., 1983), especially lipid-derived esters (Mattheis et al.,1995; Yahia et al., 1990). Sensory analysis of CA-stored apples revealed that intensityof fruity and floral descriptors decreased after 10 weeks in CA, while sourness andastringency were higher compared to apples stored in air. Some recovery of aromawas noted after removal from CA to air (Plotto et al., 1999). CA storage alsoincreased certain volatiles in tomato compared to air-stored fruit (Crouzet et al.,1986). Therefore, certain packaged fresh-cut products may require active modifica-tion of the atmosphere so as to insure desirable flavor at time of consumption.

High CO2, low O2 refrigerated CA storage is most likely associated with decreasedester volatile production, because respiration and the tricarboxylic acid cycle (TCA)are downregulated. The precursors to the alcohol and carboxylic acid moieties forester formation in apples are thought to originate from fatty acid and amino acidmetabolism during oxygen-dependent events (Hansen et al., 1992). Esterification ofalcohols to the corresponding acetates proceeds via oxygen-dependent AAT, in thepresence of acetyl CoA. AAT activity was suppressed by low O2 (0.5–1.0%) CAstorage in apples (Fellman et al., 1993a). Unlike melons, amino acids, which are theputative precursors for some esters, decrease during apple ripening and remain relativelyconstant in storage (Ackermann et al., 1992). Subsequently, the synthesis of impor-tant amino acids derived from the TCA and their backbone moieties will also bereduced (Brackmann et al., 1993), because reduced catabolic activity and β-oxidationlimit the substrates (mainly acetyl CoA and amino acids) required to continuouslyproduce aroma volatiles.

For fresh-cuts, the method of storage is more likely to include packaging that cancreate a modified atmosphere (MA) and possibly edible coatings that can enhancethe MA with reduced O2 and elevated CO2 levels, similar to that of CA. Use ofedible coatings has also been shown to affect flavor and levels of volatile flavorcompounds in intact citrus (Baldwin et al., 1995b; Cohen et al., 1990), apple (Saftner,1999; Saftner et al., 1999) and mango fruit (Baldwin et al., 1999b). This was likelydue to anaerobic respiration, which induces synthesis of ethanol and acetaldehyde,and to entrapment of volatiles, including ethanol and acetaldehyde, by the coatingbarrier (Baldwin et al., 1995c, 1999b). Entrapment of volatiles may be desirable incut fruit, because many will be otherwise lost due to off-gassing at the cut surface.Fresh-cut ‘Gala’ apples packaged in film pouches stored for up to 14 days lostvolatiles including farnescene, hexyl hexanoate and 2-methylbutyl hexanoate,whereas hexyl acetate and hexane increased during storage (Bett et al., 2001).

MAP is widely used for fresh-cut vegetables and fruits; however, occasionally,undesirable atmospheres can reduce quality due to discoloration and off-flavors inducedby anaerobic respiration (Mateos et al., 1993; Kader, 1986). Broccoli is particularlysensitive to MAP, creating sulfur-containing volatiles, including methanethiol and

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dimethyl disulfide in anaerobic conditions (Dan et al., 1997) which could be prob-lematic in fresh-cut florets. Foul odors were detected in broccoli florets packagedin MAP after one to three days at 5°C when O2 concentrations dropped below 1%(Ballantyne et al., 1988b); yet, CA (6% CO2 + 2% O2) at 4°C maintained flavor quality(Bastrash et al., 1993). Sweet off-odors were detected in fresh-cut lettuce stored at5°C under MAP (5% CO2 + 5% O2) by day 14 (Ballantyne et al., 1988a). Flavor losswas greatest and off-flavors were detected in fresh-cut lettuce stored at 5°C in polyeth-ylene bags by day 10, and sweet odors were detected by 17 days in vacuum-sealed PEbags (Heimdal et al., 1995). Off-odors were detected in shredded cabbage stored underCA (0–15% CO2 + 6% O2) after six days at 5°C, and when product was stored inMA pouches (with 1.7% O2), these off-odors were detected after just four days ofstorage (Kaji et al., 1993). Because fruit and vegetable tolerance to reduced O2 andelevated CO2 levels is mainly attributed to skin resistance to gas diffusion (Park et al.,1993; Theologis and Laties, 1982), a reexamination of optimum atmospheres inaddition to physical and chemical treatments for fresh-cut fruits is underway.

Most fresh-cut apple research has focused on browning, and scant sensory andflavor analysis has been performed. In fresh-cut ‘Gala’ apples packaged in film pouches,flavor intensity increased the first few days after preparation and packaging, thendissipated after five to nine days (Bett et al., 2001). ‘Gala’ apples stored in CA had adecrease in volatile production as well as a decrease in fruity flavor (Plotto et al., 1999).We evaluated postharvest and flavor changes in browning-inhibited (BI = 2 or 4%sodium erythorbate + 0.1% calcium chloride) stored fresh-cut ‘Gala’ apples preparedimmediately after harvest (pre-CA) or after CA storage (three months, 1.4% CO2 and3% O2 + one month refrigeration at 4°C). All pre-CA Hunter L* values were higher thanpost-CA for all treatments on all sampling days, and both BI treatments maintainedcolor for 14 days (Figure 12.3). In general, wedge color was superior in most pre-CAvs. post-CA treatments throughout the 14 days of storage. Although BI treatmentsmaintained color, most wedges stored in linear low-density polyethylene (LLDPE)pouches or air generally experienced short-term ester increase, followed by a significantdecrease and concomitant alcohol increases. Most recovered alcohols (e.g., ethanol, 1-butanol, 1-hexanol and 1-octanol) increased significantly after two days of storage inpouches [Figure 12.4(a)], whereas there were slight decreases in alcohol concentrationin flow-through air. Most flavor-related esters generally increased by day two anddecreased continuously if held in pouches or decreased until day seven then graduallyincreased through 14 days when stored in flow-through containers [Figure 12.4(b)].With one exception (hexyl 2-methylbutanoate), BI-treated tissue stored in pouchesgenerally had lower ester recovery after 14 days. Accumulation of esters in LLDPEpouches could be explained by a respiratory- or wound-induced burst associated withdecreasing resistance for volatiles escaping the tissue, then general decline with ensuingcatabolism of pyruvic acid into alcohols, as opposed to the TCA.

Numerous ester volatiles potentially having flavor impact in apples (e.g., butylacetate, 2-methyl-1-butyl acetate, pentyl acetate, butyl hexanoate, hexyl acetate andbutyl hexanoate) followed the trend illustrated in Figure 12.4(b), as pouchesapproached anaerobic conditions (data not shown). On the other hand, CA-storedfresh-cut ‘Bounty’ peach wedges stored in either air flow-through or CA atmosphere(1% O2 + 5% CO2) had extremely similar flavor volatile profiles. In preliminary

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fresh-cut peach experiments, the concentration of linalool (3,6 dimethyl-1,6-octa-dien-3-ol) increased on day two in tree-ripe (TR = harvested at 29.7 N, processedat < 26.6 N) wedges but declined in commercially ripe (CR = firmer, less mature)wedges [Figure 12.5(a)]. The concentration of most characteristic flavor lactones(e.g., γ-decalactone) increased after two days of storage in wedges prepared fromboth TR and overripe (OR) fruit but declined faster in OR as compared to TR throughseven or 12 days of storage, respectively [Figure 12.5(b)]. Poly-packed (MA) fresh-cut fruits may suffer substantial flavor loss after roughly one week of storage if filmsdo not have adequate CO2 and O2 transmission rates or if fruits were previously CA-stored, especially if fresh-cut product is temperature abused.

FLAVOR LIFE VS. SHELF LIFE

Flavor and aroma qualities are most often the true indicators of shelf life from theconsumer’s point of view. Unfortunately, “quality” of intact vegetables and fruits gen-erally emphasizes maintenance of appearance, at times sacrificing flavor and texture(Sapers et al., 1997). Much variability exists in the fresh-cut literature regarding accept-ability based on sensory evaluations, and this variability can often be attributed todifferent varied sensory analyses and experimental design. For example, sensoryevaluation determined that fresh-cut honeydew, kiwi, papaya, pineapple and canta-loupe stored at 4°C were unacceptable after 7, 4, 2, ∼7 and 4 days, respectively(O’Connor-Shaw et al., 1994). Yet, fruit were not sanitized and gloves were not wornduring preparation, and subsequently, microbial decay and associated texture lossmost likely limited postcutting life. Fresh-cut pineapple stored at 4°C had excellent

FIGURE 12.3 Color change in pre-CA and post-CA (three months in 1.4% CO2 and 3%O2 at 4°C) and RA (one month in air at 4°C) stored, browning-inhibited (2% Na-erythorbate+ 0.1% CaCl2), fresh-cut ‘Gala’ apple wedges (n = 30 ± standard deviation).

80

Prepared after harvest (pre-CA)

Freshly cut

Hun

ter

L* v

alue

Stored fresh-cut 4% Na-Er 2% Na-Er

Prepared after CA and RA storage

75

70

65

60

Days stored (4°C)

0 2 7 714 140 02 7 142 0 7 142

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Flavor and Aroma of Fresh-cut Fruits and Vegetables 413

visual appearance. However, chunks in the lower portion of containers developedoff-flavors associated with microbial fermentation after seven to 10 days of storage(Spanier et al., 1998). Cantaloupe pieces stored at 2°C in ready-to-serve tray-packswere visually acceptable after 19 days, but flavor scores were low after 13 days(Silva et al., 1987). Fresh-cut honeydew melon stored in air at 5°C for six days werejudged by an informal taste panel as being flat in flavor and lacking textural char-acteristics (Qi et al., 1998). Fresh-cut orange segments that had acceptable appear-ance after 14 days of storage were found to have unacceptable flavor quality afteronly five days of storage at 4°C (Rocha et al., 1995). Likewise, undesirable flavor wasthe limiting subjective factor in sliced, wrapped watermelon stored seven days at 5°C,even though aroma was still acceptable and microbial populations were not prob-lematic until after eight days (Abbey et al., 1988).

An adequate postcutting subjective appraisal indicating acceptable postharvestquality does not necessarily imply that a product has satisfactory flavor quality.

FIGURE 12.4 Typical alcohol (a)(1-hexanol) and ester (b)(2-methyl-1-buytl acetate) volatileprofiles (SPME, GC-MS) for fresh-cut ‘Gala’ wedges ± browning inhibitor (2% Na-erythor-bate + 0.1% CaCl2) stored at 4°C in LLDPE pouches or flow-through air (n = 3 ± standarddeviation).

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Numerous fresh-cut articles are gradually accumulating evidence suggesting thatflavor quality is normally compromised before visual quality. However, establishingoverall shelf life limits for fresh-cut fruit while taking flavor quality into consider-ation is difficult, because initial product variability (e.g., seasonal or regional),potential postcutting treatments and/or packaging affect flavor attributes differently.Furthermore, little work has been performed to assess what effect storage tempera-ture has upon volatile production in fresh-cuts. Subsequently, uniform flavor qualityand consumer acceptance of fresh-cut fruits based on aroma and flavor remain achallenging area for the industry. Fresh-cut flavor quality has recently become anarea of active research, and the industry needs to focus attention here as well.

CONCLUSION AND FUTURE RESEARCH

There remains a market niche for almost all value-added products in today’s society,and therefore, production, marketing and research on fresh-cut fruits and vegetablesare expanding. This chapter illustrates numerous areas of active research geared towardoptimizing product quality and improving consumer acceptability of fresh-cut fruit

FIGURE 12.5 Preliminary SPME, GC-MS volatile profiles (a) (linalool) and (b) (gamma-decalactone) for tree-ripe or overripe fresh-cut ‘Bounty’ peaches stored in Juice Catcher (JC)or high-profile (HP) deli containers under air or CA (1% O2 + 5% CO2) at 4°C (n = 2).

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products. Researchers and the industry need to work together to overcome barriersthat hamper national delivery of high-quality fresh-cut fruits throughout the year. Asthe foodservice industry and home meal replacement industry expand, there will be agreater demand for fresh-cut fruits and vegetables with acceptable flavor quality.

A fair amount of work has been performed concerning unveiling important fruitflavors and probable enzymatic pathways. Throughout this chapter, numerousenzymes have been mentioned that have critical roles regarding genesis of specificvolatile flavor compounds or classes of compounds. Relatively speaking, little workhas been completed concerning characterizing the important pathways and optimumenzyme conditions favoring production of desired volatile compounds. Likewise,little work has been performed to limit enzymatic production of undesirable aromacompounds. This is an area open for active research, especially because cuttingproducts exacerbates respiration and secondary volatile production and may lead tofurther volatile loss or change.

Researchers and fresh-cut producers are becoming aware that flavor quality willbecome a major driving force within the fresh-cut industry. Seed companies are lookingfor means by which to increase their profits by having more participation in associatedagricultural activities or industries. No longer will selling seed suffice. Rather, brandname products and patent rights may become important. With this in mind, moreemphasis will likely be directed toward determining critical fresh-cut flavor volatilesand precursor compounds and elucidating their biosynthetic pathways for enzymaticregulation. A successful fresh-cut market, especially fruits, may indeed be the drivingforce required to breed flavor back into some of our important fruits and vegetables.

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Abbey S.D., E.K. Heaton, D.A. Golden, and L.A. Beuchat. 1988. “Microbiological andsensory quality changes in unwrapped and wrapped sliced watermelon.” J. Food Prot.51:531–533.

Ackermann J., M. Fischer, and R. Amado. 1992. “Changes in sugars, acids, and amino acidsduring ripening and storage of apples (Cv. Glockenapfel).” J. Agric. Food Chem.40(7):1131–1134.

Anderson J.M. 1989. “Membrane-derived fatty acids as precursors to second messengers.”In: W.F. Boss and D.J. Morre (eds.), Second Messengers in Plant Growth and Devel-opment. New York: Alan R. Liss, pp. 181–212.

Anonymous. 2000. “Supplying quality cantaloupe for fresh-cut processing.” Fresh Cut. (Jan-uary):6–12.

Baker R.A. and R.D. Hagenmaier. 1997. “Reduction of fluid loss from grapefruit segmentswith wax microemulsion coatings.” J. Food Sci. 62(4):789–792.

Baldwin E.A. 2002. “Fruit and vegetable flavor.” In: K.C. Gross, C.Y. Wang, and M.E. Saltveit(eds.), The Commercial Storage of Fruits, Vegetables, and Florist and Nursery Stocks.Washington D.C.: Agricultural Research Service.

Baldwin E.A., M.O. Nisperos-Carriedo, and R.A. Baker. 1995a. “Edible coatings for lightlyprocessed fruits and vegetables.” HortSci. 30:35–38.

Baldwin E.A., M.O. Nisperos-Carriedo, P.E. Shaw, and J.K. Burns. 1995b. “Effect of coatingsand prolonged storage conditions on fresh orange flavor volatiles, degrees Brix, andascorbic acid levels.” J. Agric. Food Chem. 43:1321–1331.

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Baldwin E.A., J.W. Scott, and R.L. Shewfelt. 1995c. “Quality of ripened mutant and transgenictomato cultigens.” Proc. Tomato Quality Workshop. 503:47–57.

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Evaluating Sensory Quality of Fresh-cut Fruits and Vegetables

Karen L. Bett

CONTENTS

IntroductionFlavorTexture Conducting Descriptive Sensory Analysis

Room Design Serving Protocol

Factors That Affect Fresh-cut Sensory Quality SummaryReferences

INTRODUCTION

Lifestyle changes in the new millennium necessitate portable, safe, nutritious andhigh-quality fresh-cut produce. Because people want the fresh fruits and vegetablesin their diet, but do not choose to prepare it, the market niche exists. Consistent fresh-like quality is what will keep consumers purchasing fresh-cut produce. Normally,flavor changes occur before the visual appearance deteriorates (Anonymous, 2000).Therefore, consumers have to feel confident they will be purchasing fresh-cut producewith good sensory quality for repeat purchasing. For the continued growth of thisindustry, flavor and texture changes have to be understood, and the quality must bemonitored. Sensory evaluation is critical to understanding and monitoring flavor andtexture changes. Such analysis can be expensive, but it is essential to understand whatis happening in the mouth before correlation with instrumental methods should beattempted (Muños et al., 1991). Sensory evaluation can be used for quality assuranceand quality control purposes, but instrumental methods, if available, are more eco-nomical on a routine basis. Consumer-based evaluations are subjective and dependon the pool of consumers being tested. Unless consumers are selected properly, thetest can be biased (i.e., company employees do not represent the population as a whole).

13

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Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Consumer or affective tests do not give information about the perceived difference(Poste et al., 1991). This chapter will focus on descriptive sensory analysis of flavorand texture, which is more effective for measuring quality changes than usinguntrained consumers. ASTM (1992) manual MNL 13 describes various methods ofdescriptive analysis. The purpose of this chapter is to give an overview of what isinvolved in sensory evaluation of fresh-cut produce. This is by no means a compre-hensive discussion on training and operating a sensory panel, but it will aid in theunderstanding of what is involved.

FLAVOR

Flavors of various fruits and vegetables are unique. An apple is distinctly differentfrom an orange. Some flavor characteristics are, however, common among fruitsand/or vegetables. Green, sweet, sour, bitter and astringent are common character-istics in most fruits and vegetables (Tables 13.1 and 13.2). Flavors common in manyfruits are caramelized/honey, chemical, estery or fruity and floral/perfumy. Theseflavors are defined in Table 13.3. Off-flavors common in fruits are deteriorated/rottenand fermented. Earthy/musty is less common. There are flavors that are unique tocertain fruits. For example, there is a melon flavor in cantaloupe, apple flavor inapples and pineapple flavor in pineapple. We have sometimes observed the appear-ance of noncharacteristic fruit flavors in apples, and pumpkin and cucumber flavorsin cantaloupe. This can occur because compounds that make up these flavors arepresent in other fruits and vegetables at different concentrations. Variations in cli-matic conditions, storage temperatures and maturity levels are examples of condi-tions that can result in the production of the atypical flavor observed. Pumpkin orcucumber flavors have been observed in immature cantaloupe but are less commonin mature fruit (Bett, 2000, unpublished data).

Typically, vegetables are not considered sweet, with a few exceptions, such ascarrots and sweet potatoes, but most have low-intensity sweet taste. Broccoli andcabbage have a strong sulfury note. Radishes have a pungent mouthfeel, and onionshave an alliaceous aroma. Carrots are sweet and have a clove flavor. Sweet taste isused as a descriptor for each vegetable in Table 13.2, and green/grassy is used todescribe several vegetables. Some attributes listed in Table 13.2 may occur in morevegetables than are recorded. Vegetables can have the off-flavor, deteriorated/rotten(not listed in Table 13.2), but it was noted in several fruits listed in Table 13.1.

Flavor intensity is the strength that a particular flavor has within a food. Intensitycan be measured on a universal intensity scale, product-specific scales or attribute-specific scales. Universal scales have several advantages. A group of trained panelistscan use the same scale for any fruits or vegetables, processed or raw. They can evaluateother products as well. In addition, one can literally compare apples and oranges(based on intensity of common descriptors). The universal scale needs to be broadenough to cover the full range of attribute intensities but discrete enough to pick upsmall differences in intensities. Universal scale references are food products thatconsistently exhibit specific flavor intensities. Many different foods can be used inone universal scale. Meilgaard et al. (1999) lists many commercially available foodsthat can be used as intensity references, along with the flavor and its intensity for

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Evaluatin

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ry Qu

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-cut Fru

its and

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TABLE 13.1Common Flavors and Tastes Reported in Fruit-Type Produce

Blueberry Cherry Grape Strawberry Orange Apple Pear Apricot Peach Plum PapayaPassion

Fruit Pineapple

Astringent x x x x x x x x x x

1

x xBitter x x x x x x x x x x x xCaramelized/honey x x x x x x x x x Chemical x x x x x x x x x xDeteriorated/rotten x x x x x x x x x x x xEstery x x x x x x x x x x x xEtheral x x x x x x x x x xFermented x x x x x x x x x x xFloral/perfumy x x x x x x x x x x x xGreen x x x x x x x x x x xSour x x x x x x x x x x xSweet x x x x x x x x x x xWoody x x x x x x x x Musty/earthy x x x x x Dried fruit x x x Citrus x x Artificial

2

x x x x x x x x xRaw x x x x x x x x Fruity x x x x x x x

1

Blank spaces mean the descriptor was not reported in this fruit in ASTM (1996) but could possibly be found in it.

2

Not recommended as a descriptor, because it may be covered in the intensity rating of another descriptor.

Source:

Adapted with permission from DS-66,

Aroma and Flavor Lexicon for Sensory Evaluation

, copyright 1996, American Society of Testing and Materials (ASTM).

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TABLE 13.2Common Flavors and Tastes Reported in Vegetables-Type Produce

Broccoli Cabbage Carrot Celery Cucumber OnionBell

Pepper Radish SpinachSummer Squash Tomato

Astringent

1

x x x xBitter x x x x x x xEarthy/musty x x x x x xFloral xGreen/grassy x x x x x x x xSalty x x x x xSour x x x xSweet x x x x x x x x x x xSulfur/sulfide x x xUmami x xFermented x xMetallic x x x xBrown caramel/caramelized x x x x xWoody x xButtery x xTobacco/paprika x xViney x xBurn x x

1

Blank spaces mean the descriptor was not reported in this vegetables in ASTM (1996) but could possibly be found in it.

Source:

Adapted with permission from DS-66,

Aroma and Flavor Lexicon for Sensory Evaluation

, copyright 1996, American Society of Testing andMaterials (ASTM).

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Evaluating Sensory Quality of Fresh-cut Fruits and Vegetables

431

TABLE 13.3List of Descriptors with Definitions and References for Flavor, Taste and Mouthfeel of Fruits and Vegetables

Alcohol

—aromatic characteristic of the chemical class of compounds known as alcohols. Ref: ethanol.

Alliaceous

—reminiscent of garlic or onion, associated with chemical family of compounds allyls. Ref: methyl allyl trisulfide at 0.05% in water or oil (aroma only and prepare under a hood).

Apple

—aromatic characteristic of different apple varieties. Ref: apple concentrate to which no essence has been added.

Astringent

—the chemical feeling factor on the tongue or other skin surfaces of the oral cavity described as puckering/dry and associated with tannins or alum. Ref: 1% alum in water.

Barny/barnyard

—aromatic characteristic of barn or barnyard

,

combination of manure, urine, moldy hay, feed, livestock odors. Ref: Tincture of Civet, full strength.

Bell pepper

—aromatic associated with green bell peppers. Ref: 0.01 ppm 2-isobutyl-3-methoxy pyrazine.

Bite

—chemical burning, sensation felt on tongue or in the mouth and throat. Ref: 0.15% red pepper in water.

Bitter

—taste on tongue stimulated by solutions of caffeine, quinine and certain other alkaloids. Ref: 0.1% solution of caffeine or quinine.

Burn/heat

—chemical feeling factor associated with high concentrations of irritants to the mucous membranes of the oral cavity. Ref: 30% alcohol solution, 10% NaCl solution, white vinegar.

Buttery/diacetyl

—aromatic associated with artificial butter. Ref: 0.5 ppm diacetyl in water.

Cabbage, raw

—sweet, earthy aromatics associated with raw cabbage. Ref: fresh, washed cabbage.

Caramelized/browned caramel

—sweet aromatic characteristic of browned sugars and other carbohydrates. Ref: caramelized sugar.

Cardboardy

—aromatic associated with slightly oxidized fats and oils, reminiscent of wet cardboard packaging. Ref: malonaldehyde or wet cardboard or paper filters.

Chemical

—a very general term associated with many different types of compounds, such as solvents, cleaning compounds and hydrocarbons. Ref: product specific, bleach, caustic cleaning solutions.

Citrus

—aromatic associated with general impression of citrus fruits. Ref: citrus oils.

Cloves

—a sweet, brown-spice, almost minty aromatic associated with cloves. Ref: eugenol.

Cucumber

—flavor typical of fresh cucumber. Ref: raw cucumber.

Deteriorated/rotten

—aroma associated with rotten, deteriorated, decayed fruit/material. Ref: rotten fruits (specific).

Dried fruit

—flavor associated with dried fruits. Ref: dried apples, apricots, peaches, pears, prunes, figs.

Earthy/musty

—aromatic characteristic of damp soil, wet foliage or slightly undercooked boiled potato. Ref: 0.3

µ

g geosmin/L water, 30 ppb 2-methyl isoborneol or 0.4 ppm alpha fenchol in water.

Estery

—ripe fruit character associated with esters. Ref: 0.5 ppm ethyl butyrate in water or other fruity compound.

Etheral

—flavor associated with chloroform. Ref: 5 ppm acetal in water.

Fermented

—aromatic associated with fermented fruits or vegetables. Ref: fermented apple juice or WONF 3RA654 (McCormick).

Floral

—a sweet, fragrant aromatic associated with flowers. Ref: rose oil, 1 ppm geranoil in water, 2 ppm phenyl ethyl alcohol in water.

Fruity

—aromatic associated with a mixture of nonspecific fruits (berries, apples/pears, tropical, melons, usually not citrus). Ref: 2 ppm ethyl butyrate, ethyl caprilate and ethyl acetate.

(

continued

)

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the Spectrum Intensity scale for descriptive analysis. Unstructured line scales (6 inchesor 15 cm) that usually have an anchor on each end have the advantage over numericalscales in that no steps or “favorite” numbers exist. Panelists’ repeatability can be moredifficult to attain, because the position on the line is not stored in memory like thenumber in a numerical scale. The type of scale used is decided by the panel leader,taking into account management’s goals.

TABLE 13.3List of Descriptors with Definitions and References for Flavor, Tasteand Mouthfeel of Fruits and Vegetables (Continued)

Green apple

—the aromatic associated with freshly harvested green varieties of apples. Ref: 5.0 ppm

trans

-2-hexen-1-al.

Green/grassy

—aromatic characteristic of freshly cut leaves, grass or green vegetables. Ref: 50 ppm

cis

-3-hexen-1-ol.

Metallic

—a flat chemical feeling factor stimulated on the tongue by metals. Ref: 0.1–1% ferrous sulfate solution in water.

Moldy

—aromatic characteristic of mold growth or mildew. Ref: 10,000 ppm 2-ethyl-1-hexanol in glycol.

Onion

—aromatic associated with onion. Ref: 0.50 g onion powder in 200 ml water.

Overripe tomato

—aromatic associated with overripe tomatoes.

Peely/peel oil

—flavor associated with peel or skin flavor. Ref: apple peel, grape skin or orange oil.

Raw

—aromatic associated with unprocessed and/or uncooked product. Ref: fresh fruit or vegetable.

Salty

—taste on tongue stimulated by sodium salt, especially sodium chloride. Ref: 0.3% NaCl in water.

Seedy

—character associated with chewing on seeds. Ref: raspberry and blackberry seeds.

Skunky/mercaptan

—aromatic associated with sulfur compounds, which exhibit a skunk-like character. Ref: 0.1% furfuryl mercaptan in alcohol and water.

Soapy

—aroma associated with unscented soap. Ref: deconoic or dodeconic acid, Ivory Snow flakes.

Sour

—basic taste on tongue stimulated by acids. Ref: citric acid, vinegar, lactic acid.

Sulfide, sulfur

—aromatic associated with hydrogen sulfide, rotten egg. Ref: hydrogen sulfide bubbled through water (use caution) or old eggs hard boiled.

Sweet

—taste on the tongue stimulated by sugars and high potency sweeteners. Ref: 5% sucrose in water.

Terpeny

—aromatic associated with pine volatiles. Ref: 0.05% beta-pinene or alpha-terpineol in water (sniff carefully).

Tobacco/paprika

—aromatic associated with paprika. Ref: paprika.

Tomato

—a general term that combines those characteristics commonly associated with tomato. Ref: 2–5 ppm methional.

Umami/monosodium glutamate

—specific chemical feeling factor stimulated by monosodium glutamate (MSG). Ref: 0.2% solution of MSG.

Viney

—aromatic associated with green wood/small young branch or stems of plants. Ref: alpha- lonone on perfumer’s stick.

Waxy

—aromatic reminiscent of waxes. Ref: gamma-undecalactone on perfumer’s stick.

Woody

—aromatic associated with dry, fresh-cut wood; balsamic or bark-like. Ref: 10 ppb alpha-humulene in water or alcohol or wood chips.

Source:

Adapted with permission from DS-66,

Aroma and Flavor Lexicon for Sensory Evaluation

,copyright 1996, American Society of Testing and Materials (ASTM).

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TEXTURE

Texture, as opposed to flavor, is the structure and orientation of the food and thereaction of the food to an applied force. As in flavor characteristics, the varioustexture attributes are labeled and defined, similar to flavor characteristics. The textureattributes of produce can be divided into four areas: surface properties, first biteproperties, chew down and after swallowing properties (Meilgaard et al., 1999).Table 13.4 lists the textural properties with definitions with the high- and low-intensityreferences common to most fruits and vegetables. Surface moisture (wetness) androughness are surface properties. Springiness, cohesiveness, denseness, hardness,moisture release, juiciness, crispness and uniformity of bite are all determined onthe first bite. Chewiness and cohesiveness of mass are determined during chew down.Mouth coating is evaluated after swallowing.

The texture intensity scales are unique for each attribute. Many of the attributescales for the spectrum method are published in Meilgaard et al. (1999). Some ofthe overall most important attributes are crispness, hardness and juiciness or moisturerelease (which measure virtually the same attribute). Crispiness is important in somefruits or vegetables, because it indicates the degree of turgor pressure within the cellsor the amount of wilting that has taken place. Hardness indicates the senescencethat has occurred. Juiciness or moisture release can be an indication of the amountof dehydration that has occurred. The moisture release scale was developed atSouthern Regional Research Center (SRRC). The intensities included in Table 13.4 arerepresentative of the low and high ends of the scale. These universal scales areapplicable for all foods that have these characteristics. For number scales, panelistsare encouraged to use tenths to help define differences between fruit. Therefore, scalesspecific for fruits and vegetables are not needed.

CONDUCTING DESCRIPTIVE SENSORY ANALYSIS

R

OOM

D

ESIGN

The sensory laboratory should be set up specifically for the purpose of evaluatingproduce samples. The lab should be accessible but away from congested areas wherenoise levels can be loud. If panelists are drawn from people outside the work site,the laboratory should be near an entrance but away from machine shops, productionlines or cafeteria kitchens, where odors may interfere with sensory work. If employ-ees are used as panelists, a good location would be to have the panelists pass by thesensory laboratory on their way to the cafeteria or break room. The test facility shouldconsist of booths for individual evaluation of samples, a conference table for trainingand a separate sample preparation room. Special booth features that enhance oper-ation include a light signal system and a data entry device (keypad, tablet digitizeror personal computer). The conference room should be large enough to accommodate

Slight Extreme

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TABLE 13.4Fruit and Vegetable Sensory Texture Descriptors

∗∗∗∗

Phase/Descriptor Definition/Reference Scale

Phase I. Hold sample to lips and pass the tongue over the surface.

Wetness The amount of moisture due to an aqueous system on the surface.

Low

=

unsalted soda cracker

==>

high

=

Oscar Mayer ham luncheon meat

Roughness The amount of particles in the surface.Low

=

gelatin dessert

==>

high

=

Finn Crisp rye wafer

Phase II. Evaluate before or at first bite.

Cohesiveness The degree to which sample deforms rather than crumbles, cracks or breaks.

Low

=

corn muffin

==>

high

=

sun-dried seedless raisinsCrispness The force and noise with which a product breaks or fractures

when compressed with the molar teeth.Low

=

Quaker Low Fat Chewy Chunk granola bar

==>

high

=

Pepperidge Farm Goldfish cheese crackerHardness The force to attain a given deformation (force to compress

between molars).Low

=

cream cheese

==>

high

=

LifeSavers hard candyJuiciness

**

The amount of juice/moisture perceived in the mouth.Low

=

banana

==>

high

=

watermelonMoisture release

**

Amount of wetness/juiciness released from the sample.Low

=

(Betty Crocker) Gusher

==>

high

=

grapeDenseness The compactness of the cross section.

Low

=

General Foods Cool Whip topping

==>

high

=

Farley Fruit slices

Phase III. Evaluate during chewing.

Cohesiveness of mass The degree to which a chewed sample holds together in a mass.

Low

=

shoestring licorice

==>

high

=

archway Cookies soft brownie

Chewiness The amount of work to chew the sample.Count the number of chews required to prepare for swallowing.

Phase IV. Evaluate after swallowing.

Mouth coating The amount of residue left in the mouth and on teeth surfaces after expectorating or swallowing.

Low

=

cooked cornstarch

==>

high

=

tooth powder (any brand)

*

Note:

Most of these descriptors are in Meilgaard et al. (1999).

**

These two descriptors are similar, but the latter was developed at SRRC, and theformer was from Meilgaard et al. (1999).

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all panelists (10–15 people). A conference table (or tables arranged in conferencestyle) that seats a full panel and “electronic write board” are needed. Low-pressuresodium lamps, colored bulbs or theater gel filters over recessed lights can be usedto mask color differences. The preparation room for fresh-cut produce should consistof a separate room from the booths and training room. If processing is done in aplant or food preparation laboratory, then the basic requirements of counter space,sink and a refrigeration unit would be sufficient. For more detailed information, referto ASTM (1986) and ISO (1988) guidelines. Booths and conference room shouldbe held at 22–24

°

C (72–75

°

F) and 45–55% R.H. A slight positive pressure shouldbe maintained to prevent odors from coming from surrounding facilities or thepreparation laboratory into the booth area.

S

ERVING

P

ROTOCOL

Fresh-cut fruits and vegetables are cut into bite-size pieces and placed in 0.178 liters(6 oz) glass custard cups and covered with 125 mm watch glasses. Avoid custardcups with fluted rims to prevent loss of volatiles. Watch glasses allow the headspaceto trap volatiles for sniffing. Refrigerated samples should be allowed to warm up toambient temperature before being presented to the panelists to allow more volatilesto emanate. It is of utmost importance that

all

samples be served at the sametemperature. The same number of approximately the same size cubes should beplaced in each custard cup. A new plastic fork that does not emit an aroma or aclean metal fork is used for tasting each sample. The presentation order should berandomized to minimize order bias. The first sample should be a nonexperimental,warm-up sample to help standardize the panel and minimize position effect on thefirst sample. Panelists should smell the sample first and then evaluate oral flavorand/or texture. Texture has a logical order. Surface properties are evaluated first,followed by first bite, then chew down and last, the properties measured afterswallowing. Flavor has an order, also. Some tastes such as sweet and salty and someflavor notes are perceived early, while others are perceived later.

FACTORS THAT AFFECT FRESH-CUTSENSORY QUALITY

Cultivar differences can have significant effects on flavor and texture. Cultural andenvironmental conditions (climate, fertilizer application, soil conditions) can alsocause changes in a given cultivar that could affect flavor and texture. Some cultivarsare typically sweeter, have a more intense characteristic flavor or a softer texture. Somecultivars retain quality during fresh-cut storage better than others. Gorny et al. (1999)observed 13 cultivars of peaches during fresh-cut storage and found that ‘Flavorcrest,’‘Elegant Lady,’ ‘Red Cal’ and ‘Cal Red’ retained quality during fresh-cut storage longerthan nine other cultivars. This work focused on visual appearance and not flavor andtexture. In unpublished data (Bett, 2000, unpublished data), in two years of cantaloupecultivar comparisons at SRRC during fresh-cut storage, ‘Pacstart’ got harder and‘Athena’ got softer one year and remained the same another year. Kim et al. (1993)

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reported that firmness of fresh-cut apples decreases with storage, but ‘Golden Deli-cious’ apples remained firm longer than ‘Monroe,’ ‘Rhode Island Greening’ and ‘NewYork 674.’ Some cultivars endure fresh-cut storage better than others. Plant breedersare developing new breeding lines that focus on fresh-cut processing quality.

Crop maturity has a significant impact on quality of fresh-cut produce. Gornyet al. (1998) reported that overripe stone fruit had a short shelf life (two days forpeaches and three to six days for nectarines), while mature-green and partially ripefruit increases in firmness during fresh-cut storage. Fresh-cut mature cantaloupeevaluated at SRRC retained its fruity flavor, while the immature cantaloupe fruitincreased in fruity intensity with storage. Immature fruit tend to get harder duringstorage than mature fruit. Fruit maturity affects fruit quality, significantly (Watadaand Qi, 1999). Immature fruit lack good sensory quality, and over-mature fruit havelimited shelf life capacity.

Processing environment is critical to flavor and texture quality as well as tominimizing microbial problems. The temperature of the processing room should bemaintained at 2.8

°

C. Contact surfaces need to be routinely cleaned and sanitized.Personnel need to use clean gloves, clean aprons, hair covers and surgical masks(Sargent, 1998). Substandard processing conditions can contribute to heavy micro-bial loads that lead to quality deterioration as well as possible safety problems.

Cutting the produce typically causes the respiration rate to increase. Therefore,minimal cutting is better for shelf life because it keeps the respiration nearer to thatof intact produce (Watada et al., 1996). Removal of stems from grapes and hullsfrom strawberries changes the respiration rate less than peeling and slicing apples.Ideally, produce should be processed enough to make it convenient, but with minimaltissue damage.

Sanitizers can impart a flavor on produce if allowed to remain on the surfaces.Chlorine has a familiar characteristic odor, hydrogen peroxide and ultraviolet raysimpart little to no flavor or aroma changes. Ozone has a faint ozone aroma, but itdissipates quickly. Peroxyacetic acid has a mild acetic acid/vinegar flavor. Irradiationhas been proposed as a means of extending shelf life, but in whole fruits and vegetables,the doses required for microbial spoilage prevention cause tissue softening (Maxieet al., 1971).

Browning of cut surfaces can be a problem in some produce. Browning in cutfruit is usually caused by oxidation of phenols catalyzed by polyphenol oxidaseenzymes. Browning in apples can increase the sweet aroma or caramel flavor. Ittypically can be prevented in fresh fruit by coating cut surfaces with a browninginhibitor, which in some cases, could impart a different flavor to the produce. Sodiumerythorbate and sodium lactate, for example, impart a salty taste on the surface.Calcium chloride or calcium lactate can impart a slight bitter taste, but also, canmake the product slightly firmer (Agar et al., 1999; Luna-Guzmán and Barrett, 2000).

Packaging normally creates an atmosphere to maintain conditions that increaseproduct storage time. Permeability of the film can be selected to retard or enhancechlorophyll degradation, control browning, delay mold development and retard growthof some microorganisms (Cartaxo et al., 1997; Senesi et al., 1999; Watada, 1997).All of these can affect the sensory properties of the fresh-cut product. Selecting theappropriate packaging material can control the relative humidity surrounding the fruit

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that can prevent dehydration of tissue and control condensation (Watada, 1997; Watadaand Qi, 1999), both of which cause deterioration in sensory quality.

Proper storage temperature is critical for microbial quality, but it also affectssensory quality. Elevated temperature hastens processes such as respiration, browningand microbial growth. Microbial growth can generate off-flavors such as fermented,deteriorated/rotten and moldy and can become more intense at a faster rate withhigher temperatures (Abbey et al., 1988). The increased enzymatic activity that occursat higher temperatures accelerates browning and tissue softening (Watada, 1997).Storage temperatures that are too low can cause chilling injury. Chilling injuryweakens tissues, because they are unable to carry on normal metabolic processes.After chilling injury, produce may look sound, but upon warming up slightly, theydevelop symptoms such as pitting, skin blemishes, internal discoloration or failureto ripen. Temperatures that cause a slight amount of chilling injury are preferredover temperatures that cause rapid senescence and microbial deterioration (Watadaand Qi, 1999).

SUMMARY

Flavor and texture of fresh-cut produce are critical to consumer satisfaction. Sensoryquality can be affected at every step from production to storage conditions at thepoint of consumption, and descriptive analysis is an effective tool to measure andmonitor these differences. Produce optimization needs to be considered at each pointin the production, processing and distribution chain. When sensory evaluation isimplemented on fresh-cut produce, it must be carried out under very controlled andconsistent conditions. This chapter has given an overview of what is involved indescriptive sensory analysis and how different aspects of the processing and distri-bution chain can affect sensory properties.

REFERENCES

Abbey, S.D., Heaton, E.K., Golden, D.A. and Buechat, L.R. 1988. “Microbial and sensory qualitychanges in unwrapped and wrapped sliced watermelon.”

J. Food Prot.

51:531

533.Agar, I.T., Hess-Pierce, B. and Kadar, A.A. 1999. “Postharvest CO

2

and ethylene productionand quality maintenance of fresh-cut kiwi fruit slices.”

J. Food Sci.

64:433

−440.Anonymous. 2000. “No substitute for flavor in fresh-cut.” Fresh Cut. May: 22−23, 28.ASTM. 1986. Physical Requirements Guidelines for Sensory Evaluation Laboratories. J.

Eggert and K. Zook, Eds. ASTM Special Technical Pub. 913. American Society forTesting Materials, Philadelphia, PA.

ASTM. 1992. Manual on Descriptive Analysis Testing for Sensory Evaluation. R.C. Hootman,Ed. ASTM Manual Series MNL 13. American Society for Testing Materials, Phila-delphia, PA.

ASTM. 1996. Aroma and Flavor Lexicon for Sensory Evaluation. G.V. Civille and B.G. Lyon,Eds. ASTM Data Series Publication DS 66. American Society for Testing Materials,West Conshohocken, PA.

Bett, K.L. 2000. Unpublished data. Southern Regional Research Center, U.S. Department ofAgriculture, Agricultural Research Service, New Orleans, LA.

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438 Fresh-cut Fruits and Vegetables: Science, Technology, and Market

Cartaxo, C.B.C., Sargent, S.A., Huber, D.J. and Lin, C.M. 1997. “Controlled atmospherestorage suppresses microbial growth on fresh-cut watermelon.” Proc. Fla. State Hort.Soc. 110:252−257.

Gorny, J.R., Hess-Pierce, B. and Kader, A.A. 1998. “Effects of fruit ripeness and storagetemperature on the deterioration rate of fresh-cut peach nectarine slices.” Hort Sci.33(1):110−113.

Gorny, J.R., Hess-Pierce, B. and Kader, A.A. 1999. “Quality changes in fresh-cut peach andnectarine slices as affected by cultivar, storage atmosphere and chemical treatments.”J. Food Sci. 64(3):429−432.

ISO. 1988. Sensory Analysis—General Guidelines for the Design of Test Rooms. InternationalStandard ISO 8587. International Organization for Standardization, 1 rue Varembé,CH-1211 Génève 20, Switzerland.

Kim, D.M., Smith, N.L. and Lee, C.Y. 1993. “Quality of minimally processed apple slicesfrom selected cultivars.” J. Food Sci. 58(5):1115−1117, 1175.

Luna-Guzmán, I. and Barrett, D.M. 2000. “Comparison of calcium chloride and calciumlactate effectiveness in maintaining shelf stability and quality of fresh-cut canta-loupes.” Postharvest Biol. Technol. 19:61−72.

Maxie, E.C., Sommer, N.F. and Mitchell, F.G. 1971. “Infeasibility of irradiating fresh fruitsand vegetables.” Hort Sci. 6:202−204.

Meilgaard, M., Civille, G.V. and Carr, B.T. 1999. Sensory Evaluation Techniques, ThirdEdition. CRC Press, Boca Raton, FL, pp. 195−208.

Muños, A.M., Civille, G.V. and Carr, B.T. 1991. Sensory Evaluation in Quality Control. VanNostrand Reinhold, New York, NY, p. 20.

Poste, L.M., Mackie, D.A., Butler, G. and Larmond, E. 1991. Laboratory Methods for SensoryAnalysis of Food. Canada Communications Group-Publishing Centre, Ottawa, Canada,pp. 61−68.

Sargent, S.A. 1998. “Fresh-cut watermelon—New guidelines take cutting out of producedepartment’s backroom.” Citrus and Vegetable Magazine, Feb.:26−28, 44.

Senesi, E., Galvis, A. and Fumagalli, G. 1999. “Quality indexes and internal atmosphere ofpackaged fresh-cut pears (Abate Fetel and Kaiser varieties).” Ital. J. Food Sci.11(2):111−120.

Watada, A.E. 1997. “Quality maintenance of fresh-cut fruits and vegetables.” Foods andBiotechnol. 6(4):229−233.

Watada, A.E. and Qi, L. 1999. “Quality of fresh-cut produce.” Postharvest Biol. Technol.15:201−205.

Watada, A.E., Ko, N.P. and Minot, D.A. 1996. “Factors affecting quality of fresh-cut horti-cultural products.” Postharvest Biol. Technol. 9:115−125.

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Future Economic and Marketing Considerations

Greg Pompelli

CONTENTS

BackgroundOpportunity CostsCharacteristics of Agricultural IndustriesConsumers

Forces Shaping Fresh-cut Produce MarketsTechnology AdoptionFresh-cut Produce ConsumersMarket DevelopmentsCompetitive Pressures

Future Supply Chain Management ConsiderationsSummaryReference

BACKGROUND

The food-processing industry offers consumers a new convenience-oriented foodproduct almost every day of the year (

Progressive Grocer

, 1999). Given the high costof development and marketing, companies launching these products must believetheir products will be well received by consumers who indicate their lives are madebetter by the added convenience incorporated in the goods they purchase. Most ofthe chapters in this book focus on the development of processes and materials thatallow companies to convert highly perishable raw agricultural commodities intodesirable branded products. The great story of the fresh-cut industry is its ability tomatch technological know-how to consumer wants. Unfortunately, with so manyfirms attempting the same strategy, providing added convenience is not a sufficientguarantee of success. Value, or benefits divided by price, is a critical element indetermining a product’s success with consumers, just as it is with wholesale andinstitutional buyers.

14

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The assessment of value occurs all along the fresh-cut supply chain. As firmssearch for ways to provide desired attributes (e.g., ease-of-use or retained nutritionalvalue) using new and established technologies, they have recognized the need to ensurequality and uniformity in their inputs. The application of a new membrane technology,for example, is useless if the produce going into the bag is poorly handled or stressedby pest damage or other natural pressures. The contest for consumers’ food expendi-tures, both at-home and away-from-home, is a circular process of assessment that,depending on where one wishes to begin, starts in the field and ends with consumers,or starts with consumers and ends in the field. In the first case, analysts follow physicalproducts, and in the latter case, they follow the dollars used as economic signals.

This chapter briefly outlines economic and market factors that the fresh-cutproduce industry may face as it matures and strives to grow. An essential part of theindustry’s future growth and presence in the food market will depend on its abilityto deliver value to consumers, develop and implement new technologies, and rewardall participants in the supply chain. Much of the industry’s recent success is due tothe convergence of consumer awareness and needs with technological developmentsand raw product availability.

O

PPORTUNITY

C

OSTS

The industry’s future prospects will rely on continuous gains in understandingconsumer needs, technological developments in agricultural and nonagriculturalindustries, and the actions and offerings of competing industries. The primary influ-ence of these factors on the future prospects of the fresh-cut produce industry arisesfrom a very basic economic concept called “opportunity cost.” The fresh-cut produceindustry is by no means alone in facing this influence, because opportunity costsinfluence the economic decisions of participants in every market. Opportunity costsof consumers, firms, and growers define the economic environment within whichthe fresh-cut produce industry has grown and within which it will exist.

Opportunity costs are a common link throughout this description, which startswith farm production and its declining share of consumer food dollars. The use ofnew technologies and non-farm inputs in the creation of fresh-cut products are amongthe most important reasons farmers receive a declining proportion of each consumerdollar. As consumers demand more product transformations (packaging, processing,grading, transportation, etc.), the portion of the product’s cost/price accounted forby the raw farm product diminishes. With that said, the return to growers needs tobe sufficient to keep resources in fruit and vegetable production. Each season,growers must compare their fruit or vegetable returns to the next best (most profit-able) enterprises that they could operate using the resources they employ in theirfruit or vegetable operations.

C

HARACTERISTICS

OF

A

GRICULTURAL

I

NDUSTRIES

One complicating factor that makes agricultural production unique among industriesis the reliance on natural, often uncontrollable inputs. Production of fruits and veg-etables represents a combination of biological and physical processes that are affectedby input use decisions, weather, and other natural pressures. To varying degrees,

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Future Economic and Marketing Considerations

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production is dependent on germination and biological processes that occur withinspecific seasons and over extended periods of time within the seasons but not underentirely certain conditions. Extended time lags, years in the case of some perennialcrops, exist between production decisions and product harvests. Furthermore, whencompared to manufacturing industries, agricultural production decisions are not nearlyas closely linked to harvests or output decisions. As a result, producers make theirdecisions based, in part, on expectations about growing conditions, prices, and futuremarket access. The use of expectations and the lagged supply response can lead toprice and production cycles over time.

The intensive and extensive use of natural resources (e.g., land and water) playsan important role in production decisions. Historically, few uses existed for theseresources outside of agricultural production. However, as rural/urban interfacesexpand, alternative uses of these resources increase (subdivisions, manufacturing,etc.). In turn, the number of alternative uses, and the returns to those alternatives,may increase. While land resources have only slowly shifted away from agriculturaluses, capital and labor face significant alternative opportunities. Returns from fruitand vegetable production for land, labor, and capital must be as attractive as thenext best alternative, or those factors of production will shift to other industries.

A further complicating factor is that fluctuating supplies and prices lead to fluc-tuating producer incomes. In response to income risk, producers may diversify theirproduction mix so that stable or increasing prices for one set of products might offsetfalling prices for other products. However, diversification reduces specialization, andthat can lead to higher average costs. For all but the largest U.S. farms, diversificationcan, and often does, include off-farm income. The potential for off-farm employmentraises the opportunity cost labor used to produce fruits and vegetables, not only becauseof the income stream, but also because of potential benefits such as health coverage.

Seasonal overproduction is especially difficult to offset for perishable fruits andvegetables. The lack of market alternatives combined with inelastic demand has aheavily downward pressure on prices. Even growers with honored contracts mayreceive lower prices in the long-run if overproduction persists. As prices fall, so dothe marginal product values of resources, and without offsetting technological inno-vations or reallocation of resources, farm incomes decline.

Opportunity costs influence producers’ resource use, production mix, and farmand off-farm employment decisions. In turn, growers evaluate these alternatives todecide what and how much to produce. Growers make these assessments by com-paring the expected returns to the “next” best rate of return their resources couldreceive in another use. The comparison may be viewed from long- or short-termperspectives, but they nonetheless occur.

C

ONSUMERS

At the other end of the marketing chain, opportunity costs influence consumers asthey maximize their “utility” by making choices about purchases given limited bud-gets, a host of household characteristics, and an ever-changing set of tastes and prefer-ences. Opportunity costs come into play for consumers as they assess the expectedbenefits or utility derived from one basket of goods compared to another set of goods.

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The satisfaction consumers derive from consumption is typically a function ofincome, background, lifestyle, family composition, and nutritional need factors. Asa result, the assessment made by one consumer may be different from that of anothergiven differing household or personal characteristics.

FORCES SHAPING FRESH-CUT PRODUCE MARKETS

T

ECHNOLOGY

A

DOPTION

One reason the assessment of opportunity costs is a constant process in the fresh-cut produce industry is that many forces affect or may affect the industry. Theseforces include changes in consumer demand, technological developments that enablefirms to deliver desired product qualities, the application of new food technologiesin other industries, industry consolidation across the supply chain, and changes inthe regulatory environment.

As is the case for most industries, processing and marketing firms lead thedevelopment and utilization of new technologies. Not surprisingly, technologicaldevelopments and market consolidation forces are often complementary. The costsassociated with technological developments and resulting gains in output reduce thenumber of firms that can profitably operate in a specific market. Technology is asource of the economies of size that provide market advantages for larger firms.Technology also helps differentiate goods in the marketplace. Brands that use specifictechnologies may become known for fresher, greener, or safer products. Those char-acteristics become the basis on which brands become differentiated.

Important consequences of the adoption of new technologies are often the needfor more consistent raw inputs and consolidation in the technology-adopting industry.Consistent product standards are required to fully exploit new technologies. The needsfor consistent product deliveries and product standards affect the way in which growersproduce fruits and vegetables and leads to greater reliance on contracts over open markettransactions. Consolidation leads to fewer marketing alternatives for growers (e.g.,Fresh Express, Inc. and Dole Fresh Vegetables, Inc. control over 70% of the packagedsalad market). Fewer marketing choices often limit opportunities for price discovery,which limits market participation and increases the importance of contracts.

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Almost two decades of rising U.S. per capita consumption of fresh fruits and vege-tables represent a wonderful example of suppliers reading the consumers’ signals. Asshown in Chapter 1, changing dietary habits, hectic lifestyles, increased raw productavailability, and an expanding selection of fresh-cut fruits and vegetables have madethe fresh-cut produce market the fastest growing in the fruit and vegetable industry.In short, the rapid growth of the fresh-cut produce industry shows what can happenwhen convenience and variety foster consumers’ good intentions, and the technologycan be applied to meet those needs. Fresh-cut fruits and vegetables are excellent exam-ples of products that offer as much convenience as they offer nutritional value.

Nonetheless, the fresh-cut produce industry is no different from most other indus-tries. The industry bases its success on consumer and foodservice demand for its

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products. Consumers, as households or individuals, represent a combination of needsand income. Foodservice demand derives from consumer demand for meal replace-ments both at- and away-from-home. Both foodservice and consumers look for newor unique products, convenience in preparation/use, and price. The influence of thesewants on the market is weighted by consumer incomes that determine the capacityto purchase goods. The wants and ability to pay determine “effective” demand.

While income is a primary force driving changes in demand for fresh-cut products,demographic, lifestyle, workforce patterns, and health considerations also influencedemand. Income is the element that effectively transforms wants and needs into effec-tive demand. Demographic factors (e.g., age, gender, education, ethnic background, andhousehold composition) significantly influence consumer wants. Single-parent house-holds possess different product characteristic needs compared to seniors.

Factors such as a lifestyles and values play an important role in forming consumerwants. Consumer profiles often categorize consumers using predominantly lifestyleor value terms such as, “Strivers,” “Adapters,” and “Achievers,” because consumerconsumption patterns are so heavily influenced by lifestyles and attitudes. Under-standing the effects of these factors is especially important, as they are expected tobe drivers of future consumer/foodservice trends and fresh-cut technological devel-opment. For example, as the U.S. population ages, older age groups are expected todesire products that offer greater dietary benefits or increased convenience.

Marketers can use these profiles to develop products to include desired character-istics behavior, such as greater convenience or health benefits. However, firms do notcreate these products unless the expected return is sufficient to warrant the investmentand resource use needed to bring the product to market. The assessments used to makeproduction decisions are based on expectations about consumer behavior and arelimited by actual and anticipated technological developments. Although firms rarelyknow the opportunity costs associated with production decisions with certainty, theyare forced to allocate their scarce resources/inputs among different business activities.

Convenience and nutrition will remain significant demand factors that will affectthe characteristics of the products the fresh-cut produce industry delivers. Althoughmost consumers may not know much about their next meal, except that it should beready in less than 30 minutes, consumers are concerned about more than justconvenience and ease of preparation. Nutritional value is important, but freshness,shelf life, packaging, availability, and functional properties, not the least of whichis taste, heavily influence purchase decisions.

While characteristics, such as freshness, nutrition, and functional properties, areconsidered important, they are often only important to the extent that consumers aremade aware of their importance. Consumers gather information from a variety ofsources, including family, friends, healthcare providers, magazines, web-based sources,advertising, etc. The variety and availability of sources is good but may lead to consumerburnout on health and nutrition issues. To the extent that these sources identify newproducts or health/lifestyle concepts, the fresh-cut industry will need to work withthese sources and listen to their changing messages.

Finally, variety, appearance, and taste remain essential influences on humandiets. Health claims and convenience can only go so far to guide food preferences.Taste and appearance ultimately determine consumer appeal. Favorite foods that can

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be reformulated to include other desired characteristics frequently become the “new”favorite foods. Consumers may wish to control fat and caloric intake or increasenutritional value, but they also want to enjoy their meals.

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EVELOPMENTS

Marketing serves as the primary means for assessing the ability of firms to meetconsumer desires. Part of a firm’s ability to meet consumer needs is based on itsability to collect information about consumer desires and match the consumers’willingness to pay for characteristics with the cost associated with providing thosecharacteristics. Firms combine market information and their resources and technol-ogy to determine if they could expect sufficient returns to develop a product.

However, firms must also examine their ability to reach consumers throughmarketing channels, such as distributors and retailers. Although direct marketingopportunities exist, food distributors and retailers generally control access to con-sumers. Firms also face competitors who are also trying to meet consumer needswith their products and product lines.

One of the reasons so much attention has been given to the fresh-cut produceindustry has to do with its ability to provide products consumers desire and withthe changes in industry structure. This has led to increased diversity in compositionand form of products available. Another reason for this interest relates to the extentto which goods differ from original commodities and the impact of technology.

Firms within the fresh-cut industry have shown a keen ability to meet and evenanticipate growth to date. Part of this success is due to technological developmentsand applications that made it feasible to meet consumers’ and foodservice needs. Anequally critical element was the availability of produce that met industry needs. Futuresuccess may be more difficult to attain as the fresh-cut industry grows. Technologyand research expenses, both farm-level and processing, favor larger operations. Largeroperations spread development and investment costs over larger volumes. To theextent that human stomachs/demands do not grow with industry, increased outputreduces the need for firms and farms. Thus, one of the forces affecting the maturingfresh-cut industry may well be profitability related to structural change in the numberand size of firms and farms in the industry.

Competition from traditional fresh-cut substitutes as well as new food offeringsshould motivate continued innovation by processors and growers. Given that thenovelty of products such as bagged salads has and will continue to diminish, much ofthe competitive pressure will coincide with pressures to lower processing costs,improve quality, and increase product differentiation. These pressures will be enhancedby retailers through market power and their ability to control access to consumers.

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Competitive pressures will also change grower-processor and processor-supplierrelationships. Changes in these relationships reflect the fact that concentrationheightens the importance of business ties. In open markets, suppliers offer theirgoods to any buyer willing to meet their asking or going prices, which are most likely

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known by all participants. Concentration may reduce the transaction costs associatedwith finding buyers on open markets, but it amplifies the importance of business rela-tionships and contracts and masks market information. As concentration increases,market access of producers to participants further along the supply chain (e.g.,wholesalers, retailers, and consumers) typically decreases.

Perhaps the most important force facing the fresh-cut industry will be the evolu-tion of business relationships between growers and processors because of the obviousdisparities in market power due to their relative sizes. The development of theserelationships will be explored a bit further in the supply chain management section.However, before moving on to the next section, it is important to note a few othersignificant forces in the fresh-cut market.

Most industry participants understand the effects of income distribution, life-styles, demographic changes, health concerns, and workforce changes on demandas well as they understand the effects of technological development and adoptionon supply responses. The product safety, regulatory, and brand image issues willlikely become as important as other demand and technological considerations. Ifconsumers, retailers, or foodservice buyers question the safety or quality of theindustry’s products or the reputation of individual brands, the industry’s growth willbe hampered. While product conversion efficiency and increased product appealinitiated and initially sustained industry growth, the industry has already shiftedfrom a production-driven system to a demand-driven system in which end users havecome to expect more value-added traits and higher quality.

FUTURE SUPPLY CHAIN MANAGEMENT CONSIDERATIONS

The development and maintenance of supply chains involves the integration of stepsrequired to provide the products that end users demand. Product specification, timeli-ness, quality, availability, and the minimization of total cost are all elements ofmodern supply chains. Functioning chains offer integrated management of materialsand products from input sources to final consumers and minimize the time neededto convert inputs into goods. When successful, this “team” approach satisfies con-sumer demand and creates a competitive advantage for those in the chain.

The fresh-cut industry is no different from other manufacturing industries in thatfresh-cut firms want to reduce inventory, spoilage, transportation, and distributioncosts. Fresh-cut firms also compete based on their ability to match efficiently andaccurately product characteristics with consumer preferences, and supply chainpractices aid in this response. The operative considerations for the adoption of supplychain practices are the improved ability to meet changing consumer desires, theminimization of costs across the whole supply chain, and the provision of adequatereturns to all participants in the chain.

The difficulty for growers is that supply chains generally serve the interests ofthe supplied much better than the interests of suppliers (growers). When processorsapply supply chain practices, they typically start by seeking consistent inputs thatpossess specific product attributes that can be delivered as needed. Input consistency

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is valued, because it improves processing efficiency, and delivery timing reducesinventory costs. Increased efficiency and reduced inventories also allow processorsto focus on the “core” marketing elements of their business that help them determineconsumer preferences and willingness to pay. The desire for improved raw productquality and delivery means that processors are less willing to sort, store, or conditionfruits and vegetables and more likely to insist that growers provide these services ifthey want to continue supplying products. The shift to demand-driven supply chainsmeans that growers will feel increased pressure to create traits in their produce thatgenerate premium value for the firms and users of their products. Product specifica-tion, quality, and, in part, food safety will be heavily influenced by growers’ decisions.Their influence on these traits will not, however, mean that growers always benefitfrom commercial market appeal or that their produce will receive premiums for thedesired traits. As a result, a critical force within the fresh-cut industry is the state ofgrower-processor relationships and the degree to which risks and rewards are shared.

If the fresh-cut industry is going to continue to grow, then supply chain manage-ment practices should benefit all participants in the chain. However, the extent towhich benefits are distributed is a function of the extent to which information andrisks are shared across the chain. If information and risks are not shared, then pro-cessors will gain greater market power, and growers will face fewer marketing oppor-tunities, narrower profit margins, and less control over their production practices.

There are many reasons why fresh-cut firms might adopt supply chain practices.Some argue that these practices make firms and marketing channel participants moreefficient without regard to size. They note that the performance gap between top firmsand the food industry average is narrowing. Of course, this narrowing is also aconsequence of consolidation. Nonetheless, accurate information exchanges and rapidresponses to market changes are valuable capacities in consumer-oriented markets.

Given the natural production lags associated with fruit and vegetable production,the successful application of supply chain management practices places a greaterreliance on demand forecasts, communication, and expectations about changes inconsumer preferences. These forecasts need to be shared by input suppliers andproducers, if the ability to respond quickly is truly important. Faster response timesare important in increasingly fragmented consumer markets.

Supply chain management practices may help growth in the fresh-cut industry.However, the application of these practices may not follow the simpler lock-stepapproaches seen in manufacturing industries. First, growers’ concerns about the costof increasing quality and production flexibility need to be addressed. The cost ofincreasing produce quality can be high and is typically unpredictable. Pest pressures,weather, and other problems can undo the best plans. The investment required tomaintain or increase production flexibility may be equally prohibitive. If increasedquality and flexibility are important, then growers need to rewarded. Unfortunately,the primary reward for quality- and flexibility-oriented investments is often onlymarket access. Going back to the earlier discussion of opportunity costs, the com-parison of returns to fruit and vegetable production under these conditions may notbe sufficient to keep growers’ resources in fruit and vegetable production. This maybe especially important as domestic fruit and vegetable prices are increasinglyinfluenced by international market developments. For any given grower, the options

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for reducing production costs or expanding production choices are limited. As thefresh-cut industry moves forward, it should consider ways that it can enhanceinformation exchanges among growers and other suppliers, and when appropriate,share technology and agricultural risks.

SUMMARY

The fresh-cut produce industry has experienced an extended period of growth and hasentered a new century with justifiable expectations of continued growth and success.The focus on technology transfers and fresh-cut specific research and developmentefforts has yielded new products, new brands, improved packaging, and extended shelflife in the store and at home. Much of this growth can be traced to the industry’s abilityto focus on health and nutrition characteristics while offering consumers convenienceand product consistency. As a result, the fresh-cut industry is, in many ways, aheadof other segments in the agricultural economy in that it recognizes that productiondecisions should begin with a good understanding of end users’ (consumers and food-service) demands and preferences.

REFERENCE

Progressive Grocer

. 1999. “Produce 1999: A bumper crop of new items.” 78(10): 76.

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